Peptides and their use as inhibitors of hepatitis c virus ns3 protease

ABSTRACT

Compounds of formula (I), and pharmaceutically acceptable salts and esters thereof: (I); wherein Q, R2, X, Y and Z are as defined herein; are inhibitors of the hepatitis C virus (HCV) NS3 protease.

FIELD OF THE INVENTION

[0001] This invention relates to compounds which can act as inhibitorsof the hepatitis C virus (HCV) NS3 protease, to uses of such compoundsand to their preparation.

BACKGROUND OF THE INVENTION

[0002] The hepatitis C virus (HCV) is the major causative agent ofparenterally-transmitted and sporadic non-A, non-B hepatitis (NANB-H).Some 1% of the human population of the planet is believed to beaffected. Infection by the virus can result in chronic hepatitis andcirrhosis of the liver, and may lead to hepatocellular carcinoma.Currently no vaccine nor established, therapy exists, although partialsuccess has been achieved in a minority of cases by treatment withrecombinant interferon-α, either alone or in combination with ribavirin.There is therefore a pressing need for new and broadly-effectivetherapeutics.

[0003] Several virally-encoded enzymes are putative targets fortherapeutic intervention, including a metalloprotease (NS2-3), a serineprotease (NS3), a helicase (NS3), and an RNA-dependent RNA polymerase(NS5B). The NS3 protease is located in the N-terminal domain of the NS3protein, and is considered a prime drug target since it is responsiblefor an intramolecular cleavage at the NS3/4A site and for downstreamintermolecular processing at the NS4A/4B, NS4B/5A and NS5A/5B junctions.

[0004] Previous research has identified classes of peptides, inparticular hexapeptides, showing degrees of activity in inhibiting theNS3 protease. The aim of the present invention is to provide furthercompounds which exhibit similar, and if possible improved, activity.

[0005] Llinàs-Brunet et al (Bioorganic Medicinal Chemistry Letters 8(1998) 2719-2724) described hexapeptide inhibitors of HCV. The activityof their inhibitors could be improved when the C-terminal carboxylicacid group was “activated” as an amide. Notably, benzylamides areexemplified in this document. However, derivatization as an amide wasassociated with loss of specificity of inhibition. The authors of thisreference made no suggestion that peptides shorter than six amino acidslong could be of any utility.

SUMMARY OF THE INVENTION

[0006] The present inventors have discovered that certain peptidicphenethylamides and related compounds are effective inhibitors of NS3protease. Although not wishing to be bound by any particular theory, theinventors believe that these compounds are non-covalent inhibitors ofserine protease which may be involved in a partial bond with the serinehydroxyl group of the enzyme. This distinguishes the compounds of theinvention from compounds previously suggested as serine proteaseinhibitors and which rely on formation of a covalent bond with theserine hydroxyl group.

[0007] The inventors have found that by including a phenethylamide oranalogous group at the C-terminus the activity of hexapeptide inhibitorsmay be improved. However, in some embodiments molecules containing lessthan six amino acids retain useful activity because of the presence ofthe phenethylamide, or analogous, group at the C-terminus.

[0008] According to the present invention there is provided a compoundof Formula (I), or a pharmaceutically acceptable salt or ester thereof:

[0009] wherein Q is selected from the group consisting of:

[0010] wherein X is —CH₂— or —O—;

[0011] Y is a group of formula —C(R^(a))₂— where each R^(a) isindependently selected from hydrogen, hydroxyl, carboxylic acid, loweralkyl (such as methyl), aryl (such as phenyl), heteroaryl, aralkyl orheteroaralkyl, or the two R^(a) groups together form a cycloalkyl groupcontaining 3 to 7, preferably 3 to 5 carbon atoms;

[0012] Z is a substituted or unsubstituted aryl or heteroaryl group;

[0013] R² is a lower alkyl group, optionally substituted with one ormore fluorine atoms, or is —CH₂SH;

[0014] R³ is an optionally substituted alkyl, aryl, heteroaryl, aralkyl,or heteroaralkyl group containing from 2 to 16 carbon atoms, or togetherwith R^(c) forms a ring including the nitrogen atom which bears R^(c);

[0015] R^(c) is hydrogen or a lower alkyl group or together with R³forms a ring;

[0016] R⁴ is an alkyl, alkenyl, aralkyl, heteroaralkyl, aryl orheteroaryl group containing from 2 to 16 carbon atoms or is an acidicgroup;

[0017] R⁵ is selected from (R⁶)₂NCO—, R⁷CO—, R⁷OCO—, R⁷NHCO—, R⁷CO.CO—,R⁷S (O)₂— and R⁸ pep where “pep” is an amino acid, di- or tri- peptide;

[0018] each R⁶, independently, is selected from hydrogen and optionallysubstituted, optionally interrupted lower alkyl or lower alkenyl, aryl,heteroaryl, aralkyl or heteroaralkyl groups, or the two R⁶ takentogether form a four to seven membered ring optionally containing one ormore other heteroatoms in addition to the nitrogen atoms to which the R⁶groups are bonded;

[0019] R⁷ is an optionally substituted, optionally interrupted alkyl,alkenyl, aralkyl, heteroaralkyl, aryl or heteroaryl group containingfrom 1-18 preferably 1 to 14 carbon atoms, particularly, 1-8 carbonatoms;

[0020] R⁸ is a group of formula (R⁶)₂NCO—, R⁷CO—, R⁷OCO—, R⁷NHCO—,R⁷COCO—, and R⁷S(O)₂—;

[0021] “pep” if present is an amino acid, di, or tri peptide of formulaC—B-A;

[0022] wherein A is selected from naturally and non-naturally occurringamino acids having a hydrophobic side chain containing 1-20 carbonatoms;

[0023] B may be absent, in which case C will also be absent, but ifpresent is selected from naturally or non-naturally occurring aminoacids having a side chain which includes an acidic functionality;

[0024] C may be absent, either by itself or together with B, but ifpresent may be selected from naturally or non-naturally occurring aminoacids containing an acidic functionality;

[0025] R¹³ is a group containing up to 25 carbon atoms, 0-5 oxygenatoms, 0-3 nitrogen atoms, 0-2 sulphur atoms and up to 9 otherheteroatoms which may be the same or different;

[0026] R¹⁷ is hydrogen, a lower alkyl, lower alkenyl, aryl, heteroaryl,aralkyl, heteroaralkyl, hydroxyl, alkoxy, aryloxy, aralkoxy,heteroaralkoxy, thioether, sulfonyl or sulfoxide group; and

[0027] R¹⁸ is a group containing up to 25 carbon atoms, 0-5 oxygenatoms, 0-3 nitrogen atoms, 0-2 sulphur atoms and up to 9 otherheteroatoms which may be the same or different.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Definitions

[0029] In the discussion of the invention which follows certain termsare used repeatedly. Therefore, we seek to define each at the outset.Where definitions in the text differ from those given here it should beunderstood that the possibilities set out are those which are preferredamong the broader definitions set out here.

[0030] By “lower alkyl” and “lower alkoxy” are intended groups havingfrom 1 to 10, preferably 1 to 6, most preferably 1 to 4 carbon atoms.“Lower alkenyl” groups have from 2 to 10, preferably 2 to 6 carbonatoms.

[0031] The term “aryl” as used herein implies an aromatic ringoptionally fused, e.g. benzofused, with one to three cycloalkyl,aromatic, heterocyclic or heteroaromatic rings. Preferred groupscontaining a carbocyclic aromatic radical have from 6 to 14 morepreferably 6 to 10 carbon atoms. Examples of such groups include phenyland naphthyl. The term “heteroaryl” as used herein implies aheteroaromatic ring which is optionally fused with one to threecycloalkyl, aromatic, heterocyclic or heteroaromatic rings. Heteroarylgroups, in general, include a 3 to 7 membered heterocyclic aromatic ringconsisting of one or more carbon atoms and from one to four heteroatomsselected from nitrogen, oxygen and sulphur.

[0032] Lower alkyl and lower alkoxy groups as defined above may,optionally, carry one or more aryl or heteroaryl substituent. The resultis an aralkyl, heteroaralkyl, aralkyloxy or heteroaralkyloxy group.Examples of aralkyl- and heteroaralkyl groups include, but are notlimited to, those of formula:

—C_(n)H_(2n)Ar; and —C_(n)H_(2n)Het

[0033] where “Ar” designates aryl and “Het” designates heteroaryl and nis an integer of 1 to 10. Examples of aralkyloxy- andheteroaralkyloxy-groups include but are not limited to, those offormula:

—OC_(n)H_(2n)Ar; and —OC_(n)H_(2n)Het

[0034] where “n”, “Ar” and “Het” are as defined above. Preferredaralkyl, heteroaralkyl, aralkyloxy- and heteroaralkyloxy-groupsgenerally include from 2 to 20, say from 4 to 15 carbon atoms.

[0035] Optional substituents are not particularly limited but may beselected from the following list: lower alkyl or alkenyl, aryl,heteroaryl, lower alkoxy, aryloxy or aralkyloxy, heteroaryloxy orheteroaralkyloxy, amino, nitro, halo, hydroxy, carboxylic acid, acyl,formyl, sulphonamide, acylsulphonamide, ester, amide, cyano, andtrihalomethyl groups. As appropriate an optional substituent may itselfoptionally be interrupted and/or be substituted by another substituent.

[0036] Where a group is described as “optionally interrupted” it maycontain at least one of the following:

[0037] where R is hydrogen, or an alkyl, e.g. lower alkyl, alkenyl, e.g.lower alkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl group.

[0038] Where specific stereochemistries are indicated in the formulaeherein, these are the preferred stereochemistries. It should beunderstood that enantiomeric and diastereomeric forms of each moleculeare within the scope of the invention.

[0039] General

[0040] The compounds of the present invention all have in common a“C-terminal portion” of the following formula:

[0041] Of this, the fragment:

[0042] may be thought of as derived from an α-amino acid of formula:

[0043] In the compounds of the invention this amino acid is chosen to beidentical to, or to mimic, the amino acid found at the so-called P1position at the trans-cleavage sites of the natural substrates for HCVNS3 serine protease.

[0044] Thus generally, R² is a lower alkyl group, optionally substitutedwith one or more fluorine atoms, or is the side chain of cysteine(—CH₂SH). Apart from the cysteine side chain, other preferred R¹ groupsare —CH₂—CHF₂ (the side chain of difluoroaminobutyric acid), —CH₂CF₃(the side chain of trifluoroaminobutyric acid) and —CH₂CH₃ (the sidechain of aminobutyric acid).

[0045] X is —CH₂— or —O—;

[0046] Y is a group of formula —C(R^(a))₂— where each R^(a) isindependently selected from hydrogen, hydroxyl, carboxylic acid, loweralkyl (such as methyl), aryl (such as phenyl), heteroaryl aralkyl orheteroaralkyl, or the two R^(a) groups together form a cycloalkyl groupcontaining 3 to 7, preferably 3 to 5 carbon atoms.

[0047] Preferred combinations —X—Y— are —O—CH₂— and —CH₂CH₂—, of whichthe latter is the more preferred.

[0048] Z is a substituted or unsubstituted aryl or heteroaryl group.

[0049] Preferably, it is an optionally substituted phenyl group,although other preferred possibilities include optionally substitutedindolyl, thienyl, naphthyl, and pyridyl groups.

[0050] Where present, the possible substituents may be selected from awide range, such as set out above under the heading “definitions”.Particularly, preferred substituents on phenyl are the halogens,especially chlorine and fluorine, carboxylic acid (possibly in the formof an ester, e.g. lower alkyl ester), acylsulphonamide, and tetrazole.Another preferred substituent has the formula:

[0051] which, again, may be in the form of a lower alkyl ester. W isselected from —CH₂—, —O—, and —NH— and V is selected from —CH₂— and—CHR^(b)— where R^(b) is an optionally substituted lower alkyl, loweralkenyl, aralkyl, heteroaralkyl, aryl, or heteroaryl group.Alternatively W and V together form a carbon carbon double bond,preferably with a trans configuration.

[0052] In the case where the Z group is phenyl and this is substitutedwith an acidic substituent (carboxylic acid, acyl sulphonamide ortetrazole), the acidic group is preferably at the 4-position of thephenyl group (i.e. para to the remainder of the molecule. Thesubstituent —W—V—CO₂H is however preferably at the 3-position, althoughsubstitution at the 4-position is also possible. Substitution byhalogens, particularly fluorine and chlorine is preferably at theposition(s) ortho- to the rest of the molecule—i.e. at the 2- and/or6-position. Particularly preferred compounds have an acidic group(carboxylic acid, tetrazole, acylsulphonamide) at the 4-position andeither 2 fluorines (at positions 2- and 6-) or a chlorine in the2-position.

[0053] In addition to the characteristic “C-terminal portion”illustrated above at (I), the compounds of the invention include an“N-terminal portion” linked to the “C-terminal portion”. This is chosensuch that the molecule as a whole is capable of binding to the NS3protease of HCV, and in particular in the S1 binding pocket of theenzyme.

[0054] The extent to which the compounds bind to the NS3 serine proteaseis reflected by their IC₅₀s in inhibition assays, for instance asdescribed herein. Preferably, the IC₅₀ is 100 μM or less, particularly10 μm or less and, ideally 100 nM or less. However, activity in aninhibition assay should not be thought of as the sole indicator ofusefulness. A molecule having relatively low activity may be useful foranother reason—e.g. because of its small size or relative lack ofpeptidic character which render it resistant to degradation when takenorally.

[0055] It is to be understood that pharmaceutically acceptable salts andesters of the compounds described herein are within the scope of theinvention.

[0056] Examples of the compounds of the present invention fall intoseveral groups, or aspects, and these are considered in some more detailbelow.

[0057] Compounds of the First Aspect

[0058] According to a first aspect of the present invention there areprovided compounds of Formula (1):

[0059] and pharmaceutically acceptable salts and esters thereof.

[0060] In this formula, the groups R², X, Y and Z are as defined above,and the groups described above as preferred are also preferred inFormula (1).

[0061] The various other groups present in this molecule are consideredin turn below.

[0062] R³

[0063] There are generally two possibilities for R³. The first is thatit is a side chain whose distal end is free. Alternatively, togetherwith Rc it forms a ring which includes the adjacent nitrogen atom.

[0064] When R³ is a side chain it is generally an optionally substituted(e.g. by fluorine or carboxylic acid group) alkyl, aryl, heteroaryl,aralkyl or heteroaralkyl group, containing from 2 to 16 carbon atoms.Preferred R³ groups are unsubstituted hydrocarbons, in particular alkylgroups, such as the side chains of alanine (—CH₃), valine (—CH(CH₃)₂))leucine (—CH₂CH(CH₃)₂), isoleucine (—CH(CH₃)(C₂H₅)) or cyclohexylalanine

[0065] When R³ together with R^(c) form a ring including a nitrogen atomthe ring is desirably 4 to 7 membered, particularly 5-membered and maybe saturated or unsaturated. Optionally, the ring is substituted, forinstance by one or more lower alkyl, lower alkenyl, aryl, heteroaryl,aralkyl, or heteroaralkyl groups. Substituents present on adjacent ringcarbons may together form a further 4 to 7 membered ring. Other possiblesubstituents in the ring include, among others, hydroxyl, alkoxy (suchas lower alkoxy), aryloxy, heteroaryloxy, aralkoxy, heteroaralkoxythioether, aryl-, or alkyl-, sulfonyl and aryl-, or alkyl-, sulfoxidegroups.

[0066] Preferably, when R³ together with R^(c) form a ring then theamino acid residue including the ring is a proline residue of formula:

[0067] wherein R^(d) is hydrogen or, more preferably is one of thesubstituents discussed above—i.e. it is preferably selected from loweralkyl, lower alkenyl, aryl, heteroaryl, aralkyl, or heteroaralkylgroups, a hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkoxy,heteroaralkoxy, thioether, sulfonyl and sulfoxide groups. Particularly,preferred substituents are phenyl, cyclohexyl, benzyl ether, benzylthioether, and the thioethers, sulfones and sulfoxides formed withphenyl, cyclohexyl or n-propyl groups.

[0068] R^(c)

[0069] R^(c) is preferably hydrogen or, as described above may form aring with R³. Less preferably it may be a lower alkyl group, especiallymethyl.

[0070] R⁴

[0071] R⁴ is an alkyl, alkenyl, aralkyl, heteroaralkyl, aryl orheteroaryl group containing from 2 to 16 carbon atoms. Preferably it isa lower alkyl group, for instance the side chain of alanine (—CH₃),leucine (—CH₂—CH(CH₃)₂), isoleucine (—CH(CH₃)(C₂H₅)₁ or valine(—CH(CH₃)₂), a cyclopentyl or t-butyl group. Of these the side chain ofvaline is preferred. Alternatively, R⁴ may be an acidic group, e.g. acarboxyalkyl group containing from 2 to 8 carbon atoms, such as the sidechain of aspartic acid (—CH₂COOH) or, more preferably, glutamic acid(—CH₂CH₂COOH).

[0072] R⁵

[0073] R⁵ is selected from (R⁶)₂NCO—, R⁷CO—, R⁷OCO—, R⁷NHCO—, R⁷CO.CO—,R⁷S(O)₂— and R⁸ pep where “pep” is an amino acid, di- or tri- peptide.

[0074] Each R⁶, independently, is selected from hydrogen and optionallysubstituted, optionally interrupted lower alkyl or lower alkenyl, aryl,heteroaryl, aralkyl or heteroaralkyl groups, or the two R⁶ takentogether form a four to seven membered ring optionally containing one ormore other heteroatoms in addition to the nitrogen atoms to which the R⁶groups are bonded.

[0075] R⁷ is an optionally substituted, optionally interrupted alkyl,alkenyl, aralkyl, heteroaralkyl, aryl or heteroaryl group containingfrom 1-18 preferably 1 to 14 carbon atoms, particularly, 1-8 carbonatoms.

[0076] R⁸ is a group of formula (R⁶)₂NCO—, R⁷CO—, R⁷OCO—, R⁷NHCO—,R⁷COCO—, and R⁷S(O)₂—

[0077] “pep” if present is an amino acid, di, or tri peptide of formulaC—B-A

[0078] wherein A is selected from naturally and non-naturally occurringamino acids having a hydrophobic side chain containing 1-20 carbonatoms. Possibilities include leucine, methionine, isoleucine or, morepreferably, diphenylalanine.

[0079] B may be absent. If it is, then C will also be absent. Wherepresent, B is selected from naturally or non-naturally occurring aminoacids having a side chain which includes an acidic functionality.Preferred examples are glutamic and aspartic acid, with the former beingparticularly preferred.

[0080] C may be absent, either by itself or together with B. Wherepresent it may be selected from naturally or non-naturally occurringamino acids containing an acidic functionality. Aspartic acid ispreferred, although glutamic acid is another possibility.

[0081] Preferably “pep” is absent and the compounds are tripeptides.

[0082] Preferred R⁵ groups are those of formula R⁶OCO—. Preferred R⁷groups are t-butyl and isobutyl.

[0083] Compounds of this aspect of the invention each include severalasymmetric carbon atoms and they can, therefore, exist in the form ofoptically pure diastereoisomers, mixtures of diastereoisomers,diastereoisomeric racemates or mixtures of diatereoisomeric racemates.All of these possible forms are included within the scope of the presentinvention. However, it is preferred that all amino acids or amino acidanalogues have the L-configuration. Nevertheless, some deviation fromthis generalisation can be tolerated.

[0084] Specific examples of preferred compounds of this aspect of theinvention are shown in Tables 1 to 5.

[0085] Compounds of the Second Aspect

[0086] In a second aspect, the present invention is concerned withdipeptides, and pharmaceutically acceptable salts and esters thereof. Atthe C-terminus, these resemble the tri- and higher peptides describedabove. However, instead of a third amino acid (providing the side groupR⁴ in formula (1), the molecules contain a “cap”.

[0087] One preferred group of compounds includes dipeptides of thefollowing formula:

[0088] In this formula, X, Y, Z and R² are as defined above. Preferredexamples of these groups described above are also preferred in thisembodiment. In particular, X and Y are preferably both —CH₂— and R² ispreferably —CH₂CHF₂ or —CH₂SH.

[0089] R¹³ is a group containing up to 25, preferably 4-21, particularly4-16 carbon atoms and 0-5 oxygen atoms, 0-3 nitrogen atoms, 0 to 2sulphur atoms, and up to 9 other heteroatoms, such as halogen atoms)which may be the same or different. Preferred R₁₃ groups contain anacidic functionality, preferably a carboxylic acid group which may be inprodrug form —e. g. esterified.

[0090] Substituent groups R¹³ preferably include a relativelyhydrophobic portion such as a cycloalkyl, aryl, heteroaryl, aralkyl, orheteroaralkyl group each of which may, optionally, be substituted.

[0091] Preferred examples of the group R¹³ include indolines of formula:

[0092] and tetrahydroquinolines of formula:

[0093] where R¹⁵ is hydrogen, an optionally branched, optionallyinterrupted and optionally substituted lower alkyl or lower alkenylgroup, or an optionally substituted aralkyl or heteroaralkyl group andR¹⁶ is hydrogen or an optionally substituted and optionally interruptedlower alkoxy, aryloxy or heteroaryloxy group.

[0094] A preferred substituent on R¹⁵ is —CO₂H, optionally in the formof a lower alkyl ester. When R¹⁵ is an aralkyl group it is preferably anoptionally substituted benzyl or thienylmethyl group. Preferredsubstituents in the aryl group include halogens, especially chlorine,lower alkoxy (e.g. OMe) and aryloxy (e.g. PhO—) groups, cyano, andcarboxylic acid groups. Carboxylic acid groups, optionally in the formof lower alkyl esters are especially preferred.

[0095] Especially preferred R¹⁵ groups include:

[0096] Another preferred example of the group R¹³ has the followingformula:

[0097] Once again, the carboxylic acid group may be esterified forinstance as a lower alkyl ester such as a methyl ester. Alternatively,the carboxylic acid group may be amidated, e.g. as —CONH₂.

[0098] Preferred groups of this type have the formula:

[0099] Other possible R¹³ groups are listed under the heading “R³” atTable 7. Each R¹³ exemplified may be used in conjunction with any of thegroups “R₂” and “R₁” listed in the table.

[0100] Another preferred group of dipeptides includes proline, or aproline derivative at the P2 position. Compounds in this category havethe formula:

[0101] where X, Y, Z and R² are as defined above. Preferred examples ofthese groups, described above are also preferred in this embodiment. Inparticular, X and Y are preferably both —CH₂— and R² is preferablyCH₂CHF₂ or CH₂SH. Z is preferably 2,6-difluoro-4-carboxy-phenyl-.

[0102] R¹⁷ is hydrogen or more preferably is a lower alkyl, loweralkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, a hydroxyl,alkoxy, aryloxy, heteroaryloxy, aralkoxy, heteroaralkoxy thioether,sulfonyl or sulfoxide group. Particularly preferred substituents arephenyl, cyclohexyl, benzyl ether, benzyl thioether, and the thioethers,sulfones and sulfoxides formed with phenyl, cyclohexyl or n-propylgroups. Miscellaneous examples of this group can be found at table 6.

[0103] R¹⁸ is a group containing up to 25, preferably 4-21, particularly4 to 16 carbon atoms and 0 to 5 oxygen atoms, 0-3 nitrogen atoms, 0-2sulphur atoms and up to 9 other heteroatoms, such as halogen atoms,which may be the same or different.

[0104] Some examples of suitable R¹⁸ groups include the following:

[0105] where R¹⁹ is an alkyl group, preferably a lower alkyl group,including branched especially α-branched alkyl groups such as isopropylor t-butyl groups, a C₃-C₇ cycloalkyl group, or anoptionally substitutedaryl group. Preferred substituents include C₁₋₈ alkoxy, halogen or —CF₃.

[0106] Another suitable R¹⁸ group has the formula:

[0107] wherein each R^(e) is independently selected from hydrogen, loweralkyl (especially methyl), lower alkenyl, lower alkoxy, optionallysubstituted aryl, heteroaryl, aralkyl or heteroaralkyl groups (such asthose substituted with halogen, —CF₃ or lower alkyl or alkoxy groups) ortwo R^(e) taken together result in the formation of a three to sevenmembered aliphatic or aromatic ring which optionally contains at leastone heteroatom. In the case where two R^(e) taken together result in theformation of a ring containing unsaturation, especially an aromaticring, then other R^(e) may be absent.

[0108] Optionally one or more groups

[0109] may be replaced by —O—. Preferably no more than one such group isreplaced.

[0110] A preferred subclass of this group of compounds is

[0111] such as

[0112] The carboxylic acid group in any of this preferred class ofcompounds may be esterified for instance as a lower alkyl ester such asa methyl ester.

[0113] The —OH group of the carboxyl acid group may also optionally bereplaced by an —SO₂NH— group, especially by Ph-SO₂—NH—.

[0114] Examples of other groups suitable as substitiaent R¹⁸ are shownunder the heading “R₄” in Table 6. Each of the exemplified groups may beused in conjunction with any of the other groups exemplified in thetable.

[0115] Other Aspects of the Invention

[0116] According to a third aspect, the present invention provides acompound or derivative according to the first aspect, for use in anytherapeutic method, preferably for use in inhibiting the HCV NS3protease, and/or for use in treating or preventing hepatitis C or arelated condition. By “related condition” is meant a condition which isor can be caused, directly or indirectly, by the hepatitis C virus, orwith which the HCV is in any way associated.

[0117] According to a fourth aspect the present invention provides theuse of a compound or derivative according to the first aspect in themanufacture of a medicament for the treatment or prevention of hepatitisC or a related condition.

[0118] A fifth aspect of the invention provides a pharmaceuticalcomposition which includes one or more compounds or derivativesaccording to the first aspect.

[0119] The composition may also include pharmaceutically acceptableadjuvants such as carriers, buffers, stabilisers and other excipients.It may additionally include other therapeutically active agents, inparticular those of use in treating or preventing hepatitis C or relatedconditions.

[0120] The pharmaceutical composition may be in any suitable form,depending on the intended method of administration. It may for examplebe in the form of a tablet, capsule or liquid for oral administration,or of a solution or suspension for administration parenterally.

[0121] According to a sixth aspect of the invention, there is provided amethod of inhibiting HCV NS3 protease activity, and/or of treating orpreventing hepatitis C or a related condition, the method involvingadministering to a human or animal (preferably mammalian) subjectsuffering from the condition a therapeutically or prophylacticallyeffective amount of a composition according to the fourth aspect of theinvention, or of a compound or derivative according to the first aspect.“Effective amount” means an amount sufficient to cause a benefit to thesubject or at least to cause a change in the subject's condition.

[0122] The dosage rate at which the compound, derivative or compositionis administered will depend on the nature of the subject, the nature andseverity of the condition, the administration method used, etc.Appropriate values can be selected by the trained medical practitioner.Preferred daily doses of the compounds are likely to be of the order ofabout 1 to 100 mg. The compound, derivative or composition may beadministered alone or in combination with other treatments, eithersimultaneously or sequentially. It may be administered by any suitableroute, including orally, intravenously, cutaneously, subcutaneously,etc. Intravenous administration is preferred. It may be administereddirectly to a suitable site or in a manner in which it targets aparticular site, such as a certain type of cell—suitable targetingmethods are already known.

[0123] A seventh aspect of the invention provides a method ofpreparation of a pharmaceutical composition, involving admixing one ormore compounds or derivatives according to the first aspect of theinvention with one or more pharmaceutically acceptable adjuvants, and/orwith one or more other therapeutically or prophylactically activeagents.

[0124] Compounds of the present invention may be prepared by reactingprotected form of the P1 amino acid:

[0125] with a compound of formula H₂N—X—Y-Z.

[0126] The resulting compound may subsequently be extended towards theN-terminus by conventional methods of synthesis employing protectedamino acids, peptides or “capped” amino acids.

[0127] The invention provides, according to an eighth aspect, a methodas described above for preparing a compound according to the first orsecond aspect.

EXAMPLES

[0128] Embodiments of the invention are described below by way ofexample only.

[0129] The following abbreviations are used in the examples and tables:Ac: acetate; 4-AMP: 4-aminomethylpiperidine; BEMP:2-tert.-butylimino-2-diethylamino-1,3-dimethyl-perhydro-1,3,2-diazaphosphorine; Bn: benzyl; Boc:tert-butyloxycarbonyl[Me3CO(O)]; i-Boc:isobutyloxycarbonyl[Me₂CHCH₂CO(O)]; Cbz: benzyloxycarbonyl; Cha:L-cyclohexylalanine; Cpg: L-cyclopentylglycine; DCM: dichloromethane;DIEA: diisopropylethyl amine; Dif: L-diphenylalanine; DMAP:N,N-dimethylaminopyridine; DMF: dimethylformamide; DMSO:dimethylsulfoxide; EDC: 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimidehydrochloride; Et: ethyl; EtOAc: ethyl acetate; Et₂O: diethyl ether;Fmoc: fluorenylmethyloxycarbonyl; HATU:[O-7-azabenzotriazol-1-yl)-1,1,3,3- tetramethyluroniumhexafluorophosphate]; HOBt: N-hydroxybenzotriazole; KHMDS: potassiumbis(trimethylsilyl)amide; LDA: lithium diisopropyl amide; mCPBA: metachloroperoxybenzoic acid; Me: methyl; NBS: N-bromosuccineamide; NMM:N-methyl morpholine; Ph: phenyl; pip: piperidine; PPTs:pyridinium-para-toluenesulfonate; Pr: propyl; RP-HPLC: reversed phasehigh-pressure liquid chromatography; RT: retention time; TES:triethylsilane; TFA: trifluoroacetic acid; THF: tetrahydrofuran; TIPS:triisopropylsilane; TLC: thin-layer chromatography; TMEDA:N,N,N′,N′-tetramethyl-ethylendiamine; TsCl: tosyl chloride.

[0130] Nuclear magnetic resonance (NMR) spectra were recorded on aBruker AMX 300 or AMX 400 spectrometer. The chemical shifts (δ) arereported in ppm relative to internal tetramethylsilane or the residualsolvent peak.

[0131] Mass spectral data were obtained on a Perkin Elmer API 100 innegative or positive ionization mode. Organic extracts were usuallydried over sodium sulfate, the drying agent was removed by filtrationand the solvents evaporated on a rotary evaporator under reducedpressure.

[0132] Flash chromatography was carried out on silca gel according toStill's published procedure (W. C. Still et al., J. Org. Chem. 1978, 43,2923) or on Flash chromatography systems with prepacked columns (Biotagecorporation).

[0133] Preparative RP-HPLC was carried out with a Waters Delta Prep 4000separation module, equipped with a Waters 486 absorption detector. ASymmetryPrep C18 column (7 micron, 100 A, 19×150 mm, Waters) wasroutinely used. Compounds were eluted with gradients of water (solventA) and acetonitrile (solvent B) both containing 0.1% TPA (v/v) at 17mL/min.

General Scheme for Synthesis of Compounds

[0134] The compounds of the present invention were synthesized accordingto a general process illustrated in the scheme below(where PG is anamino protecting group, usually a carbamate):

[0135] Briefly, an amine H₂N—X—Y-Z—i.e. a P′ building block—(eithercommercially available or prepared as laid out in the experimentalsection), was coupled to an amino acid containing R²—i.e. a P₁ buildingblock—using well known peptide coupling techniques. After removal of thePG group by standard techniques, a compound Q can be coupled usingpeptide coupling techniques to give a compound of formula (I).Alternatively, the compound can be elongated with another amino acidusing the methods described. This deprotection and coupling procedure isrepeated until the desired compound is obtained. Different cappinggroups (as in R¹³C═O or R¹⁸C═O) were either introduced by standard amidebond formation or by acylation of the free amino group.

[0136] Generally these reactions can be carried out in solution phase orusing solid phase methodology, where the group R² is linked to the resin(see examples 19 and 27).

[0137] The detailed examples below describe the synthesis of certain“building blocks”. Those building blocks required for synthesis of thecompounds of tables 1 to 7 and whose detailed synthesis is not describedbelow are either readily commercially available, or else can bysynthesized by techniques known to the person skilled in the art andanalogous to those described below. The building blocks whose synthesisare described are referred to as P′, P₁, P₂ or P₃ building blocks byanalogy with the terminology applied to amino acids at the cleavage siteof the natural substrate for NS3.

P′ Building Blocks Example 1 Synthesis of 3-(2-aminoethyl)thiophene (1)

[0138]

[0139] 2-(3-Thienyl)ethyl alcohol (2.62 g, 20.4 mmol) was dissolved inDCM (100 mL) and cooled to O° C. After addition of triethylamine (3.10g, 30.6 mmol) and solid tosyl chloride (3.89 g, 20.4 mmol), the reactionwas stirred at room temperature overnight. DCM was evaporated, themixture was diluted with EtOAc and washed subsequently with aqueoushydrochloric acid (1 N), water, saturated sodium hydrogen carbonate andbrine. Drying and evaporation afforded an oil, which was dissolved inanhydrous DMF (20 mL). Sodium azide (2.65 g, 40.8 mmol) was added andthe mixture heated to 40° C. for 3 h. After cooling to room temperature,EtOAc was added and the solution was washed with water (4×) and brine,then dried and evaporated. The resulting oil was filtered over silicagel (100 g) with the aid of PE/EtbAc (10:1) to obtain 2-(3-thienyl)ethylazide (2.45 g, 78% ) as a colorless liquid. ¹H-NMR (CDCl₃) δ 7.3 (dd,J=2.9, 4.9 Hz, 1 H), 7.07 (m, 1 H), 6.98 (dd, J=1.2, 4.9 Hz, 1 H), 3.51(t, J=7.1 Hz, 2 H), 2.94 (t, J=7.1 Hz, 2 H).

[0140] Reduction of the azide (2.43 g, 15.86 mmol) was accomplishedusing triphenylphosphine (4.16 g, 15.86 mmol) in THF (50 mL) and water(314 mg, 17.45 mmol) (according to B. Ganem et al., J. Org. Chem. 1987,52, 5044). After stirring overnight, the THF was evaporated in vacuo,then aqueous hydrochloric acid was added. The aqueous phase was washedthoroughly with EtOAc. Sodium hydroxide was then added until a pH of 9was reached. Extraction of the solution with DCM, evaporation and drying(sodium sulfate) gave crude 1 which was purified by kugelrohrdistillation (bp 75° C., 0.5 mbar): 1.48 g (73% ), colorless liquid.¹H-NMR (CDCl₃) δ 7.27 (dd, J=2.8, 4.9 Hz, 1 H), 6.99 (bd, J=2.0 Hz, 1H), 6.95 (dd, J=1.0, 4.9 Hz, 1 H), 2.96 (t, J=6.8 Hz, 2 H), 2.78 (t,J=6.8 Hz, 2 H), 1.24 (bs, 2 H).

Example 2 Synthesis of 2-cyclopropyl-2-phenylethylamine (2)

[0141]

[0142] 1-Phenyl-1-cyclopropanecarbonitrile (1.0 g, 6.98 mmol) was addedto a stirred suspension of lithium aluminum hydride in anhydrous diethylether (25 mL). The suspension was refluxed for 2 h, then cooled to 0°C., and the excess hydride was destroyed by careful addition of water.After treatment with sodium hydroxide (1 N), the mixture was filteredand the filtrate extracted with diethyl ether. After drying of theorganic phase with and evaporation 2 (719 mg, 70% ) was obtained as aclear liquid and used without further purification. ¹H NMR (DMSO-d₆): δ7.28 (m, 4 H), 7.17 (m, 1 H), 2.70 (s, 2 H), 1.45 (bs, 2 H); 0.78 (dd,J=6.2, 3.8 Hz, 2 H); 0.67 (dd, J=6.2 Hz, 3.8 Hz, 2 H). MS: (m/z) 148(M+1)⁺.

Example 3 Synthesis of 3-(2-Aminoethyl)-1-(tert-butyl-acetyl)indole (3)

[0143]

[0144] 2.0 g of tryptamine (12.5 mmol) were dissolved in DCM (50 mL) andtreated in one portion with triethylamine (1.74 mL. 12.5 mmol, 1 eq) andtrityl chloride (3.44 g, 12.3 mmol). The resulting solution was stirredfor 24 h at room temperature while protected from light. Volatiles wereevaporated and the residue treated with aqueous HCl (1 N). The solidformed was filtered and washed with small portions of ether thendissolved in aqueous sodium hydrogen-carbonate and extracted into DCM.The organic layer was dried and evaporated to give 4.48 g (89% ) of3-(N-trityl-(2-aminoethyl))-indole, which was used without furtherpurification.

[0145] To a solution of the foregoing compound (0.5 g, 1.24 mmol) inanhydrous THF (12 mL), BEMP (0.46 mL, 1.61 mmol) andt-butyl-bromoacetate (0.24 mL, 1.61 mmol, 1.2 eq) were added dropwisevia syringe. The solution was stirred at room temperature for 48 h andthen diluted with diethyl ether, washed with water (2×) and brine. Theorganic layer was dried and evaporated and the residue purified by flashchromatography (eluent: PE/EtOAc 7:1, 50 g silica gel) affording 537 mg(85% ) of the acylated indole.

[0146] The foregoing compound (530 mg, 1.02 mmol) was deprotected withTFA/DCM/TES (5:90:5, 11 mL). After 10 min the mixture was poured intohydrochloric acid (1 N), the aqueous layer was washed with DCM anddiethyl ether and then brought to pH 9 by addition of sodium carbonate.Extraction with DCM, drying of the organic layer and evaporation gave172 mg (63% ) of 3 as a pale-yellow oil, which was used without furtherpurification. ¹H NMR (300 MHz, DMSO-d₆): δ 7.53 (d, J=7.8 Hz, 1 H); 7.27(d, J=8.2 Hz, 1 H); 7.11 (s, 1 H); 7.11 (t, J=7.1 Hz, 1 H); 7.01 (t,J=7.4 Hz, 1 H); 4.91 (s, 2 H); 2.78 (m, 4 H); 1.98 (bs, 2 H), 1.41 (s, 9H). MS (m/z) 275 (M+1)⁺.

Example 4 Synthesis of tert-Butyl-4-(2-aminoethyl)-benzoatehydrochloride (5a) and the methyl ester 5b

[0147]

[0148] α-Bromo-4-toluic acid (10 g, 46.5 mmol) was suspended in DCM andisourea 4 (18.6 g, 93 mmol) in DCM (50 mL) was added dropwise. Theresulting mixture was refluxed for 6 h, then another portion of 4 (18.6g, 46.5 mmol) was added. After refluxing the mixture overnight, it wasfiltered through a plug of silica gel, eluting with PE/EtOAc (9:1). Acolorless oil was obtained (13.4 g) after evaporation of the solvents,which contained tert-butyl-4-bromomethyl benzoate and 14% ofdiisopropylurea. This material was dissolved in DMF (50 μL) and addeddropwise during 15 min to a solution of sodium cyanide (2.84 g, 58 mmol)in water (5 mL) at 0° C. The yellow solution was stirred for 1 h at thistemperature, then water (200 mL) was added and the aqueous phase wasextracted with diethyl ether. The organic phase was washed with waterand brine, dried (sodium sulfate) and evaporated. The crude product waspurified by flash chromatography (PE/EtOAc, 92.5:7.5) to givetert-butyl-4-cyanomethyl benzoate (8.2 g, 76% ) as a colorless oil. ¹HNMR (CDCl₃) δ 8.01 (d, J=8.3 Hz, 2 H), 7.40 (d, J=8.3 Hz, 2 H), 3.82 (s,2 H), 1.61 (s, 9 H).

[0149] The foregoing compound (5.0 g, 23.6 mmol) was hydrogenated in amixture of methanol/acetic acid (4:1, v/v, 150 mL) using palladium oncharcoal (1.5 g, 10% Pd) under balloon pressure of hydrogen overnight.After removal of the catalyst by filtration, toluene (250 mL) was addedto the filtrate and the solution was evaporated in vacuo. This processwas repeated two times. The oily residue was then dissolved in toluene(150 mL) and a solution of hydrochloric acid in diethyl ether (1 M, 60mL) was added. After evaporation, an off-white solid was obtained, whichwas crystallized from chloroforme and n-heptane. Compound 5 was obtainedas a white solid (3.42 g, 56% ). Another 2.25 g (37% ) were obtainedfrom by evaporating the mother liquor and crystallizing the residue asdescribed above. ¹H NMR (DMSO-d₆) δ 8.52 (bs, 3 H), 7.93 (d, J=6.6 Hz, 2H), 7.29 (d, J=6.6 Hz, 2 H), 3.23-3.41 (bs, 4 H), 1.58 (s, 9 H).

[0150] Using identical procedures for the cyanide displacement and thehydrogenation, but methyl-α-bromotoluic ester as the starting material(obtained from the acid by esterification with trimethylsilyldiazomethane in methanol), methyl-4-(2-aminoethyl)-benzoatehydrochloride (5b) was obtained. ¹H NMR (DMSO-d₆) δ 8.18 (bs, 3 H), 7.89(d, J=6.7 Hz, 2 H), 7.40 (d, J=6.7 Hz, 2 H), 3.80 (s, 3 H), 2.89-3.10(m, 4 H).

Example 5 Synthesis of Ethyl-3-(2-aminoethyl)-cinnamate hydrochloride(6)

[0151]

[0152] 2-(3-Bromophenyl)ethyl alcohol (2.01 g, 10 mmol) was dissolved inDCM (50 mL) and cooled to O° C. After addition of triethylamine (1.52 g,15 mmol) and tosyl chloride (1.91 g, 10 mmol) the reaction was stirredat room temperature overnight. TLC indicated complete conversion of thestarting material. DCM was evaporated, the mixture was diluted withEtOAc and washed subsequently with aqueous hydrochloric acid (1 N),water, saturated sodium hydrogen carbonate and brine. Drying andevaporation afforded an oil, which was dissolved in anhydrous DMF (10mL). Sodium azide (1.3 g, 20 mmol) was added and the mixture heated to40° C. for 16 h. After cooling to room temperature, EtOAc was added andthe solution was washed with water (4×) and brine. The resulting oil wasfiltered over (100 g) with the aid of PE/EtOAc (10:1) to obtain2-(3-bromophenyl)ethyl azide (1.63 g, 72% ) as a colorless oil. ¹H-NMR(CDCl₃) δ 7.39 (m, 2 H), 7.16-7.22 (m, 2 H), 4.51 (bs, 1 H), 3.50 (d,J=6.6 Hz, 2 H), 2.83 (t, J=6.6 Hz, 2 H).

[0153] The foregoing compound (1.60 g, 7.08 mmol) was dissolved in THF(70 mL). After triphenylphosphine (1.86 g, 7.08 mmol) and water (140 mg,7.79 mmol) was added, the mixture was stirred overnight at ambienttemperature. The solvent was evaporated, then aqueous hydrochloric acidwas added. The aqueous phase was washed thoroughly with EtOAc and DCM.Sodium hydroxide was then added until a pH of 9 was reached.2-(3-bromophenyl)-ethyl amine (922 mg, 65% ) was obtained by extractionof the solution with DCM, evaporation and drying.

[0154] The foregoing amine (750 mg, 3.75 mmol) was converted to itsBoc-derivative using Boc₂O (818 mg, 3.75 mmol) and triethylamine (569mg, 5.63 mmol) in DCM (20 mL). After workup, the Boc-protected amine(742 mg, 66% ) was isolated by flash chromatography (PE/EtOAc 12:1) as acolorless oil. ¹H-NMR (CDCl₃) δ 7.39 (m, 2 H), 7.20 (m, 2 H), 4.51 (bs,1 H), 3.28 (bd, J=6.3 Hz, 2 H), 2.79 (t, J=6.8 Hz, 2 H), 1.46 (s, 9 H).

[0155] The foregoing compound (380 mg, 1.26 mmol) was dissolved inanhydrous DMF (10 mL). Palladium acetate (28 mg, 0.126 mmol),tris-(2-tolyl)phosphine (153 mg, 0.50 mmol), sodium acetate (310 mg,3.78 mmol) and ethyl acrylate (164 mg, 1.64 mmol) were added. Thesolution was heated to 120° C. under an atmosphere of argon. After 16 h,the reaction was cooled to room temperature, EtOAc was added and theresulting solution was washed with water (3×) and brine. Drying andevaporation gave an orange oil, which was purified by flashchromatography (PE/EtOAc 10:1) to give Boc-protected 6 as a colorlessoil (308 mg, 77% ). ¹H-NMR (CDCl₃) δ 7.67 (d, J=16.0 Hz, 1 H), 7.40 (d,J=7.7 Hz, 1 H), 7.35 (s, 1 H), 7.33 (t, J=7.5 Hz, 1 H), 7.22 (d, J=7.4,1 H), 6.43 (d, J=16.0 Hz, 1 H) 4.55 (bs, 1 H), 4.27 (q, J=7.1, 2 H),3.39 (bs, 2 H), 2.81 (t, J=6.9 Hz, 2 H), 1.43 (s, 9 H), 1.34 (t, J=7.1,3 H).

[0156] 120 mg (0.375 mmol) of this material were dissolved in EtOAc (2mL) and treated with a solution of hydrochloric acid in EtOAc (3 M, 2mL). After 6 h the solvents were evaporated to give 6 as an off whitesolid (89 mg, 93% ) after washing with n-pentane. ¹H-NMR (DMSO-d₆) δ8.19 (bs, 3 H), 7.62 (d, J=16.0 Hz, 1 H), 7.60 (m, 2 H), 7.37 (t, J=7.5Hz, 1 H), 7.31 (d, J=7.1, 1 H), 6.64 (d, J=16.0 Hz, 1 H), 4.18 (q,J=7.0, 2 H), 3.08 (bs, 2 H), 2.81 (bs, 2 H), 1.25 (t, J=7.0, 3 H).

Example 6 Methyl-(3-chloro-4-(2-aminoethyl))benzoate hydrochloride (7a)

[0157]

[0158] A mixture of of methyl-(3-chloro-4-methyl)-benzoate (4.90 g,26.54 mmol), NBS (4.72 g, 26.54 mmol) and dibenzoyl peroxide (291 mg,1.06 mmol) in tetrachloromethane was refluxed for 2 h. The whiteprecipitate that had formed, was removed by filtration, the filtratediluted with DCM (150 mL) and washed with cold water (3×) and brine.Drying and evaporation afforded a yellow oil (6.24 g). ¹H NMR (DMSO-d₆,400 MHz) δ 7.96 (d, J=1.7 Hz, 1 H), 7.92 (dd, J=8.0 Hz, 1.7 Hz, 1 H),7.77 (d, J=8.0 Hz, 1 H), 4.79 (s, 2 H), 3.87 (s, 3 H).

[0159] This material was added neat to a solution oftetra-n-butylammonium cyanide (6.31 g, 23.5 mmol) in anhydrousacetonitrile (24 mL) at 0° C. After 10 min the solution was diluted withDCM, and passed through a plug of neutral alumina. The eluate wasevaporated and partitioned between diethyl ether and water. The aqueousphase was extracted with diethyl ether and the combined organic phaseswashed with water and brine. Drying and evaporation gave a brown oil,which was purified by flash chromatography on silica gel (100 g,PE/EtOAc 87:13). A yellow oil was obtained which was further purified byflash chromatography on silica gel (200 g, PE/DCM 1:1) to givemethyl-(3-chloro-4-(2-cyanpethyl))benzoate as a white solid (750 mg, 15%).

[0160] To a cold (0° C.) solution of the foregoing compound (590 mg,2.81 mmol) and CoCl₂ hexahydrate (1.33 g, 5.6 mmol) in MeOH (20 mL) wasadded NaBH₄ (1.06 g, 28 mmol portionwise during 10 min). The mixture waspoured into hydrochloric acid (1 N, 60 mL) and stirred until the blackprecipitate had dissolved. The aqueous layer was made alkaline with concammonia, extracted with CHCl₃, dried, filtered and concentrated to give7a as a brown oil, which was treated with hydrogen chloride in EtOAc(3.2 M, 2 mL). After evaporation and trituation with diethyl ether, 7awas obtained as a yellow powder (420 mg, 60% ). ¹H NMR (DMSO-d₆) δ 8.09(bs, 3 H), 7.95 (d, J=1.7 Hz, 1 H), 7.89 (dd, J=1.7, 7.9 Hz, 1 H), 7.56(d, J=7.9 Hz, 1 H), 3.87 (s, 3 H), 3.099 (bs, 4 H).

Example 7 tert-Butyl-(3-chloro-4-(2-aminoethyl))benzoate hydrochloride(7b)

[0161]

[0162] The procedures used for the synthesis of compound 7b are similarto the ones described in example 6. Bromination of3-chloro-4-methyl-benzoic acid (0.5 g, 2.93 mmol) with NBS in CCl₄ (15mL) gave 4-(bromomethyl)-3-chlorobenzoic acid as a white powder (0.68 g,93% yield). ¹H NMR (CDCl₃, 400 MHz) δ 7.92 (s, 1 H), 7.87 (dd, J=7.9 Hz,1.4 Hz, 1 H), 7.70 (d, J=7.9 Hz, 1 H), 4.76 (s, 2 H).

[0163] The foregoing compound was dissolved in DMF (3 mL) and cooled to0° C. NaCN (2 equ) dissolved in 1 mL of water and 2 mL of DMF were addedto the solution and the reaction mixture was stirred for 0.5 h. Afterdiluting the mixture with ethyl acetate it was washed with aqueousammonium sulfate and brine, dried (Na₂SO₄), filtered and evaporated togive a yellow solid which was directly used in the esterification step.2-(2—Chloro-4-carboxyphenyl)-acetonitrile (0.391 g, 2 mmol) was treatedwith 2 equiv of isourea 4 in 35 mL of DCM at reflux over night. Afterevaporation of DCM, the crude was diluted in EtOAc (50 mL) and filtered.The filtrate was concentrated and purified by flash chromatography insilica gel eluting with 1% EtOAc in petroleum ether to givetert-Butyl-4-(cyanomethyl)-3-chlorobenzoate (0.24 g, 47% yield). ¹H NMR(CDCl₃, 400 MHz) δ 8.03 (d, J=1.6 Hz, 1 H), 7.93 (dd, J=8.02 Hz, J=1.62Hz, 1 H), 7.60 (d, J=8.02 Hz, 1 H), 3.89 (s, 2 H), 1.61 (s, 9 H).

[0164] The foregoing compound (0.240 g, 0.95 mmol) in MeOH (10 mL), wasreduced with CoCl₂ hexahydrate (2 equiv) as described in example 6 togive free amine 7b as a brown oil (0.140 g, 58% ) after extraction. ¹HNMR (CDCl₃, 400 MHz) δ 7.98 (s, 1 H), 7.80 (d J=7.9 Hz, 1 H), 7.25 (d,J=7.9 Hz, 1 H), 3.10-2.80 (m, 4 H), 1.53 (s, 9 H).

Example 8 Synthesis oftert.-Butyl-(4-(2-aminoethyl)-3,5-difluoro)-benzoate hydrochloride (8)

[0165]

[0166] 3,5-Difluorobenzoic acid (25 g, 0.158 mol) was dissolved in DCM(150 mL) and dioxane (50 mL). The mixture was transferred into astainless steel autoclave. Concentrated sulfuric acid (3.6 mL) wasadded, followed by isobutene (100 mL). The mixture was kept in theautoclave for 5 days. Excess isobutene and the solvents were removedunder reduced pressure, the mixture diluted with EtOAc and washed withwater, saturated aqueous sodium hydrogen carbonate and brine. Drying andevaporation gave a dark-yellow oil, which was purified by kugelrohrdistillation (80-100° C., 1.0 mbar) to givetert-butyl-3,5-difluorobenzoate (27.51, g, 81% ) as a colorless solid.

[0167] The foregoing compound (10.34 g, 48.3 mmol) was dissolved inanhydrous THF (50 mL) and added to a solution of LDA (53.2 mmol) in THF(100 mL) at −78° C. and left for 1 h at this temperature. Anhydrous DMF(4.1 mL, 53.2 mmol) was added dropwise and 30 min later acetic acid (8mL), followed by water. The reaction was brought to room temperature andtaken into EtOAc. The aqueous phase was extracted with EtOAc and thecombined organic fractions washed with brine and dried. Kugelrohrdistillation (0.2 mbar, 100° C.) after evaporation of the solvents gave9.0 g (77% ) of tert.-butyl-3,5-difluoro-4-formyl benzoate as a yellowsolid. ¹H NMR (CDCl₃) δ 10.38 (s, 1 H), 7.57 (d, J=9.0 Hz, 2 H), 1.60(s, 9 H).

[0168] The foregoing compound (16.9 g, 69.8 mmol) was added to asolution of potassium fluoride (0.93 g) and NMM (113 mL) in isopropanol(75 mL). The mixture was cooled to 0° C. and nitromethane (12.5 mL) wasadded. After 2 h at 0° C. the mixture was stirred at room temperatureovernight. Acetic anhydride (94 mL) and sodium acetate (1.9 g) wereadded and the mixture brought to 60° C. After 1 h the reaction wascooled to room temperature, diluted with EtOAc and washed successivelywith hydrochloric acid (1 N, 2×) water, aqueous sodium hydrogencarbonate(2×) and brine. Drying and evaporation gave the nitrostyrene as a brownsolid (20.5 g). ¹H NMR (CDCl₃) δ 8.14 (d, J=14.0 Hz, 1 H), 7.89 (d,J=14.0 Hz, 1 H), 7.61 (d, J=9.1 Hz, 2 H), 1.60 (s, 9 H).

[0169] The nitrostyrene was dissolved in isopropanol (215 mL) andchloroform (1 l). Silica gel (172 g) was added followed by sodiumborohydride (11.0 g, 292 mmol) in 10 portions. After 30 min TLCindicated complete consumption of the starting material. Afteracidification with hydrochloric acid (100 mL, 1 N) the silica gel wasremoved by filtration and washed thoroughly with chloroform. Thecombined filtrates were washed with water and brine, dried andevaporated.

[0170] Crystallization of the crude product from hot ethanol gave 8.9 g(43% ) of tert-butyl-3,5-difluoro-4-(2-nitroethyl) benzoate as a brownsolid. The mother liquor was evaporated and the remaining solid purifiedby flash chromatography (PE/EtOAc 18:1) to yield another 460 mg (2% ).¹H NMR (CDCl₃) δ 7.52 (app d, J=7.9 Hz, 2 H), 4.62 (t, J=7.3 Hz, 2 H),3.49 (t, J=7.3 Hz, 2 H), 1.60 (s, 9 H).

[0171] The foregoing compound (5.32 g, 18.5 mmol) was dissolved inmethanol (93 mL) and hydrogenated over palladium on charcoal (1.0 g, 10%Pd) under atmospheric pressure overnight. Filtration and evaporation inthe presence of diethyl ether containing hydrogen chloride (28 mL, 1 N)gave 8 as a yellow solid (5.3 g, 98% ). ¹H NMR (DMSO-d₆) δ 8.19 (bs, 3H), 7.51 (d, J=7.7 Hz, 2 H), 3.26 (m, 4 H), 1.57 (s, 9 H). MS (m/z) 258((M+H)⁺, free amine).

Example 9 Synthesis of 9

[0172]

[0173] The formylation of 3,5-difluorobenzonitrile (2 g, 14.4 mmol) indry THF (8 mL) was carried out as described for 3,5-difluorobenzoate inexample 8. The crude residue was purified by flash chromatography(PE/EtOAc 8:1) and afforded 1.53 g of pure aldheyde (60% ). ¹H NMR (400MHz, CDCl₃): δ 10.34 (s, 1 H); 7.30 (d, J=7.3 Hz, 2 H). MS (m/z), 168(M+1)⁺.

[0174] As described in example 8, the foregoing aldehyde was convertedto the nitrostyrene, which was reduced with sodium borohydride to give655 mg of pure 3,5 difluoro-4-(2-nitroethyl)benzonitrile. ¹H NMR (400MHz, CDCl₃): δ 7.22 (d, J=6.1 Hz, 2 H); 4.61 (t, J=7.1 Hz, 2 H); 3.42(t, J=7.1 Hz, 2 H). MS (m/z) 213 (M+1)⁺.

[0175] The foregoing compound (262 mg, 1.23 mmol) was dissolved inanhydrous DMF (5 mL), treated with ammonium chloride (132 mg, 2.47 mmol,2 eq) and sodium azide (160 mg, 2.47 mmol, 2 eq) and heated overnight at120° C. After evaporation of DMF the residue was taken up into EtOAc,washed with water, 0.1 N hydrochloric acid and brine. The organic layerwas dried and evaporated to afford 132 mg (42% ) of nitro tetrazole. ¹HNMR (400 MHz, DMSO-d₆): δ 7.73 (d, J=7.8 Hz, 2 H); 4.84 (t, J=6.8 Hz, 2H); 3.36 (t, J=6.7 Hz, 2 H).

[0176] Reduction of the nitro group with 10% Pd/C (26 mg) in methanol (5mL) at atmospheric pressure of hydrogen for 48 h gave the desired amine9 (78 mg, 67% ) as a colorless solid after removal of the catalyst byfiltration and evaporation of the solvent. ¹H NMR (400 MHz, CDCl₃) δ7.57 (d, J=8.6 Hz, 2 H); 4.61 (t, J=6.8 Hz, 2 H); 3.45 (t, J=6.7 Hz, 2H); 1.89 (bs, 2 H).

Example 10 Synthesis of 12

[0177]

Synthesis of 10

[0178] The formylation of 1,3-difluorobenzene was carried out asdescribed in example 8. 1,3-difluorobenzene (10 g, 87.65 mmol) inanhydrous THF (174 mL) was added over 5 min to a stirred solution of LDA(48.20 ml, 2M solution in hexane, 96.41 mmol) in THF (20 ml) at −78° C.After 1 h at −78° C., anhydrous DMF (7.46 ml, 96.41 mmol) was addeddropwise over 5 minutes. The reaction was further stirred at −78° C. for20 min, then quenched with glacial AcOH (15 ml) and water (200 ml) andquickly extracted with diethyl ether (3×60 ml). The combined organiclayers were washed with water and brine, dried and evaporated to afford12.3 g of 2,5-difluoro-benzaldehyde. ¹H-NMR (CDCl₃): δ 7.02 (m 2 H),7.58 (tt, J=6.2, 8.5 Hz, 1 H), 10.38 (s 1 H).

[0179] The aldehyde was dissolved in benzene (173 ml), 1,2-ethanediol(259.86 mmol, 16.13 g) and PPTs (17.3 mmol, 4.35 g) were added and themixture was refluxed for 4 h. Water was removed with a Dean-Stark trap.After evaporation of the solvent the residue was taken into diethyletherand washed with sodium hydrogen carbonate and brine, Drying andevaporation dried over afforded 15 g of crude product. A portion of thismaterial (5 g) were purified by kugelrohr distillation (0.6 mbar,150-160° C.) giving 4.7 g of pure ketal. ¹H-NMR (CDCl₃): δ 4.05 (m 2 H),4.21 (m 2 H), 6.25 (s 1 H), 6.89 (m 2 H), 7.29 (tt, J=6.25, 8.4 Hz, 1H).

[0180] TMEDA (25.68 mmol, 3.9 ml, 1.2 eq) was added to a solution ofBuLi (2.2 M in hexane, 11.67 ml, 1.2 eq) in THF (70 mL) at −78° C. Thena solution of the foregoing ketal (21.4 mmol, 3.98 g) in THF (40 mL) wasadded dropwise over 10 min. The solution was stirred for 1 h at −78° C.,then trimethylborate (64.2 mmol, 6.67 g) was added in one portion. Afterwarming to 0° C., the reaction was acidified to pH 6.5 with HCl (1M),the THF was evaporated and the residue extracted with DCM after additionof a solution of saturated ammoniumchloride. The combined organic layerswere washed with brine, dried and evaporated to give a pink solid (4.5g), which converted immediately to the phenol (according to K. S. Webband D. Levy, Tetrahedron Letters, 1995, 36, 5117).

[0181] A mixture of the foregoing compound (19.58 mmol, 4.5 g), NaOH(21.54 mmol, 0.86 g), water (60 ml) and acetone (20 ml) was stirred for5 min until complete dissolution, then sodium bicarbonate (108.86 mmol,9.14 g) was added. The mixture was cooled to 0° C., a solution of oxone(11.75 mmol, 7.2 g) in an aqueous solution of EDTA (44 ml, 0.004 M) wasadded dropwise, maintaining the internal temperature below 8° C. duringthe addition. After 30 min, the reaction was quenched with sodiumbisulfite (1 g in 10 ml of water), diluted with EtOAc and acidified withconcentrated hydrochloric acid. The aqueous phase was extracted withEtOAc, the combined organic layers were washed with distilled water,dried and evaporated to give 3.8 g of crude phenol 10. Flashchromatography (PE/EA 4:1) gave pure 10 (2.5 g, 63% ) as a colorlesssolid. ¹H-NMR (CDCl₃): δ 4.05 (m 2 H), 4.21 (m 2 H), 5.09 (s, 1 H), 6.22(s 1 H), 6.78 (dt, J=1.95, 9.5, 1 H), 6.96 (dt, J=5.19, 9.3 1 H).

Synthesis of 11

[0182] Phenol 10 (300 mg, 1.48 mmol) was dissolved in acetone (7.5 mL)containing potassium carbonate (409 mg, 2.96 mmol).Methyl-2-bromopropionate was added and the mixture was refluxed for 3 h.After cooling the reaction to room temperature, EtOAc was added, theorganic phase was washed with water and brine, dried (sodium sulfate)and evaporated. The crude product was taken into acetone (15 mL), water(4 mL) and PPTs (107 mg, 0.43 mmol) were added and the resulting mixturewas refluxed overnight. After cooling to room temperature, EtOAc wasadded, the organic phase was washed with aqueous sodiumhydrogencarbonate and brine, dried (sodium sulfate) and evaporated. Thealdehyde 11 (304 mg) was 906 pure as judged by ¹H-NMR and taken directlyto the Henry aldol reaction. ¹H-NMR (CDCl₃, 400 MHz), d 10.34 (s, 1 H),7.22 (dt, J=9.0, 5.2 Hz, 1 H), 6.89 (dt, J=1.75, 9.3 Hz, 1 H), 4.74 (q,J=6.8 Hz, 1 H), 3.77 (s, 3 H), 1.66 (d, J=6.8 Hz, 3 H).

Synthesis of 12

[0183] Aldehyde 11 (300 mg, 1.23 mmol) was reacted with nitromethane andthe resulting nitrostyrene was reduced to the nitroethyl compound asdescribed in example 8. After flash chromatography (PE/EtOAc 5:1) 196 mg(55% ) of the nitroethyl compound were obtained. This material (190 mg,0.66 mmol) was dissolved in methanol (7 mL) and hydrogenated underatmospheric pressure of hydrogen using palladium on charcoal (40 mg, 10%Pd). After filtration and evaporation amine 12 (155 mg) was obtained,which was used without further purification. ¹H-NMR (CDCl₃, 400 MHz), d6.68-6.87 (m, 2 H), 4.69 (q, J=6.9 Hz, 1 H), 3.76 (s, 3 H), 2.95 (t,J=7.1 Hz, 2 H), 2.83 (t, J=7.1 Hz, 2 H), 1.68 (d, J=6.8 Hz, 3 H). MS(m/z) 260 (M+H)⁺.

P₁ Building Blocks

[0184] (L)-2-Amino-4,4-difluorobutanoic acid (13) was synthesizedaccording to Burger et al. (Synthesis, 1996, 1419). It was converted toN-carbamate protected derivatives 14 using standard methodology, asexemplified in example 11.

[0185] Other amino acids used like 2-aminobutanoic acid or2-amino-4,4,4-trifluorobutanoic acid are commercially available.

Example 11 Synthesis of 14

[0186]

[0187] 1.0 g (7.19 mmol) of 13 was converted to its Cbz derivative usingN-(benzyloxycarbonyloxy)-succinimide (CbzOsu, 1.79 g, 7.19 mmol) in amixture of water (30 mL) and dioxane (30 mL) containing sodium carbonate(1.52 g, 14.38 mmol). After stirring overnight, dioxane was evaporatedand an extractive workup (see also example 15) yielded 2.0 g(quantitative) of 14a as a colorless oil, which was used without furtherpurification. ¹H-NMR (DMSO-d₆) δ 2.08-2.40 (m, 2 H), 4.13 (m, 1 H), 5.04(s, 2 H), 6.08 (app. tt, J=56.2, 4.7 Hz, 1 H), 7.20-7.48 (m, 5 H), 7.78(d, J=7.7 Hz, 1 H), 12.9 (bs, 1 H). MS (m/z) 274 (M+H)⁺.

[0188] From similar experiments, but using Boc-anhydride or FmocOSuinstead of CbzOSu gave 14b (88% ) or 14c (quantitative).

[0189] 14b: ¹H-NMR (DMSO-d₆ ) δ 1.37 (s, 9 H), 2.08-2.29 (m, 2 H), 4.03(app. dt, J=5.0, 8.8 Hz, 1 H), 6.07 (app. tt, J=56.4, 4.6 Hz, 1 H), 6.92(bs, 1 H), 7.25 (d, J=8.3 Hz, 2 H), 12.8 (bs, 1 H). MS (m/z) 240.5(M+H)⁺. 14c: ¹H-NMR (DMSO-d₆) δ 2.10-2.40 (m, 2 H), 4.13 (m, 1 H), 4.21(t, J=6.6 Hz, 1 H), 4.32 (d, J=6.9 Hz, 2 H), 6.07 (app. tt, J=56.2, 4.7Hz, 1 H), 7.32 (t, J=7.3, 2 H), 7.41 (t, J=7.3, 2 H), 7.70 (d, J=7.3 Hz,2 H), 7.79 (d, J=8.3 Hz, 1 H), 7.88 (d, J=7.3 Hz, 2 H), 12.9 (bs, 1 H).MS (m/z) 362 (M+H)⁺.

P₂ Building Blocks Example 12 Synthesis of(2S,4S)-Fmoc-4-cyclohexyl-pyrrolidine-2-carboxylic acid (15a) and theBoc-derivative 15b

[0190]

[0191] Commercial (2S,4S)-Boc-4-phenyl-pyrrolidine-2-carboxylic acid(1.0 g, 3.43 mmol) was deprotected using TFA (10 mL) in DCM (10 mL). Theresulting oil obtained after evaporation of the solvents andcoevaporation with hydrogen chloride in diethyl ether was dissolved inethanol (20 mL). Platinum oxide (100 mg) was added and the resultingmixture was stirred under an atmosphere of hydrogen overnight. After 3 hanother 100 mg of platinum oxide was added. Filtration and evaporationgave a colorless solid (782 mg). This compound was converted to thecarbamates as described in example 11.

[0192] 15a (mixture of rotamers): ¹H NMR (300 MHz, DMSO-d₆) δ 12.58 (bs,1 H) 7.85 (m, 2 H); 7.60 (m, 2 H); 7.20-7.41 (m, 4 H), 4.28 (m, 1 H);4.25 (m, 1 H); 4.14 (m, 2 H); 3.52 (m, 2 H); 2.92 (m, 1 H); 1.90 (m, 3H); 1.59 (m, 4 H); 1.13 (m, 4 H); 0.88 (m, 2 H). MS (m/z) 420 (M+1)⁺.15b (mixture of rotamers): ¹H NMR (300 MHz, DMSO-d₆) δ 12.44 (s, 1 H);4.09 (t, J=8.6 Hz, 1 H); 3.51 (m, 1 H); 2.85 (m, 1 H); 1.89 (m, 3 H);1.59 (m, 5 H); 1.37 and 1.32 (s, 9 H); 1.13 (m, 4 H ); 0.90 (m, 2 H).

Example 13 Synthesis of 4-(R)-cyclohexylthio-proline methyl esterhydrochloride (18)

[0193]

Synthesis of 17

[0194] DMAP (100 mg, 0.824 mmol) and p-toluenesulfonyl chloride (1.72 g,9.07 mmol) were added successively to a solution of 16 (2.02 g, 8.24mmol) in pyridine (4 ml) at 0° C. under nitrogen and the solution wasstirred at room temperature for 48 hrs. The solution was diluted withEtOAc and the organic phase washed with 1N aqueous HCl, aqueoussaturated NaHCO₃, brine, dried and evaporated to leave a residue whichwas purified by flash chromatography (PE/EtOAc) yielding 1.35 g (41% )of a colorless oil. ¹H NMR (rotameric mixture) (DMSO-d₆, 400 MHz): δ0.75-0.85 (1 H, m), 1.29 (5 H, s), 1.33 (4 H, s), 1.98-2.04 (1 H, m),2.39 (3 H, s), 3.25-3.35 (1 H, m), 3.50-3.55 (1 H, m), 3.55 (1 H, s),3.57 (2 H, s), 4.25-4.30 (1 H, m), 5.00-5.06 (1 H, m), 7.45 (2 H, d,J=8.0 Hz), 7.72 (2 H, d, J=8.0 Hz). MS (m/z) 400.1 (M+H)⁺.

[0195] To a solution of the foregoing compound (730 mg, 1.83 mmol) inEtOH (2.7 ml) at room temperature under nitrogen were added 2.5 ml of a2M solution of Na and cyclohexyl mercaptan in EtOH and the solutionstirred overnight. The solvent was evaporated in vacuo and the residuewas purified by flash chromatography (PE/EtOAc) to obtain 451 mg (70% )of 17 as a colorless oil. ¹H NMR (rotameric mixture) (DMSO-d₆, 400 MHz):δ 1.12-1.36 (17 H, m), 1.48-1.54 (1 H, m), 1.59-1.67 (2 H, m), 1.79-1.88(2 H, m), 2.07-2.19 (2 H, m), 2.70-2.76 (1 H, m), 3.09-3.14 (1 H, m),3.37-3.44 (1 H, m), 3.67-3.73 (1 H, m), 4.05-4.15 (2 H, m), 4.15-4.22 (1H, m); MS (m/z) 358 (M+H)⁺.

Synthesis of 18

[0196] To compound,17 (236 mg, 0.661 mmol) dissolved in EtOAc (3 ml)under nitrogen at room temperature was added HCl/EtOAc (2.5 ml) and thesolution stirred for 2 hrs. The solvent was removed in vacuo and theresidue washed with Et₂O to leave 18 as a colourless oil (100% ). ¹H NMR(DMSO-d₆, 400 MHz): δ 1.16-1.29 (8 H, m), 1.48-1.55 (1 H, m), 1.61-1.68(2 H, m), 1.81-1.86 (2 H, m), 2.07-2.13 (1 H, m), 2.75-2.84 (1 H, m),2.95 (1 H, dd, J=7.0, 11.6 Hz), 3.34 (m, 1 H), 3.52 (1 H, qn, J=6.9 Hz),3.64 (1 H, dd, J=7.2, 11.6 Hz), 4.18 (2 H, q, J=7.2 Hz), 4.48 (1 H, t,J=8.0 Hz); MS (m/z): 257.9. (M+H)⁺.

Example 14 Synthesis of (4R)-(3-phenyl-propyloxy)-proline (20)

[0197]

[0198] According to the procedure of Smith et al. (J. Med. Chem. 1988,31, 875), a mixture of sodium hydride (0.69 g, 60% dispersion in mineraloil, washed with hexane) and N-Boc-L-hydroxyproline (2.0 g, 8.7 mmol) in20 mL of THF was stirred at room temperature for 45 min. 1.5 equiv of1-bromo-3-phenylpropane were added and mixture was refluxed for 3 h.Reaction mixture was poured into sodium hydroxide (1 N) and the aqueouslayer was washed with hexane, acidified with HCl (1 N) and extractedwith DCM. The organic phase was dried over sodium sulfate, filtered andconcentrated to afford the title compound 20a as a colorless oil (0.5 g,17% yield) 1 H NMR (CDCl_(3,) 400 MHz) δ 7.33-7.17 (m, 5 H), 4.52-4.44(m, 1 H), 4.08-4.03 (m, 1 H), 3.54-3.39 (m, 4 H), 2.27 (t, J=7.33 Hz, 2H), 2.44-2.19 (m, 2 H), 1.94-1.85 (m, 2 H), 1.51 (s, 9 H).

[0199] The Boc group was removed with hydrogen cloride in dioxane (483mg of 20a, 7 mL, 4 M, 1 h at room temperature). After evaporation of thesolvents, the amine hydrochloride was converted to the Fmoc protectedcompound 20 using FmocOSu as described in example 11 (504 mg, 78% ). ¹HNMR (DMSO-d₆, 300 MHz) δ 7.88 (m, 2 H), 7.65 (m, 2 H), 7.42 (m, 2 H),7.32 (m, 3 H), 7.25 (m, 2 H), 7.19 (m, 2 H), 4.25-4.35 (m, 2 H),4.12-4.23 (m, 2 H), 4.06 (m, 1 H), 3.58-3.35 (m, 4 H), 2.61 (t, J=7.4Hz, 2 H), 2.29 (m, 1 H), 1.97 (m, 1 H), 1.73-1.84 (m, 2 H).

P₃ Building Blocks Example 15 Synthesis ofN-(Isobutyloxycarbonyl)-valine (21)

[0200] L-Valine (5.86 g, 50 mmol) was dissolved in a mixture of water(200 mL) containing sodium carbonate (10.6 g, 100 mmol). The solutionwas cooled to 0° C., then a solution of isobutylchloroformate (6.76 g,49.5 mmol) in dioxane (100 mL) was added dropwise over 50 min. Theice-bath was removed and the reaction stirred overnight. Dioxane wasevaporated, the aqueous phase was extracted once with diethylether andthe brought to pH 2 by addition of hydrochloric acid. Extraction withDCM (3×, 150 mL each), drying of the organic phase (sodium sulfate) andevaporation gave 21 (14.2 g) as a colorless solid. ¹H-NMR (DMSO-d₆) δ12.48 (bs, 1 H), 7.24 (d, J=8.2 Hz, 1 H), 3.95 (dd, J=6.2, 8.3 Hz, 1 H),3.73 (d, J=6.7 Hz, 1 H), 2.01 (m, 1 H), 1.83 (m, 1 H), 0.87 (bs, 12 H).

Example 16 Synthesis of N-(Isobutyloxycarbonyl)-γ-tert-butyl-glutamate(22)

[0201] Using the conditions described in example 15,(γ-O-tert.-Butyl)-L-glutamic acid (10 g, 49.2 mmol) was converted to 22(colorless solid, 14.2 g, 95% ). ¹H-NMR (DMSO-d₆) δ 12.57 (bs, 1 H),7.34 (d, J=8.1 Hz, 1 H), 3.95 (m, 1 H), 3.72 (d, J=6.6 Hz, 1 H), 2.25(m, 1 H), 1.93 (m, 1 H), 1.85-1.71 (m, 2 H), 1.38 (s, 9 H).

Example 17 Synthesis of Indoline (25)

[0202]

Synthesis of 23

[0203] O-tert-butyl-N,N′-diisopropylisourea 4 (10.55 g, 52.7 mmol) wasadded to a solution of 3-Methyl-2-thiophene-carboxylicc acid in DCM (45ml). The reaction mixture was slowly warmed to reflux and maintained atreflux for 8 hours. Another 10.55 g of 4 was added and reflux wascontinued for an additional 10 hours. Cooling, filtration andevaporation left a solid residue which was suspended in EtOAc/PE (1:9)and filtered over a short pad of silica-gel. Evaporation of the solventsgave 6.1 g (87% ) of the tert-butyl ester.

[0204] To a solution of the above compound (5.0 g , 25.4 mmol) in CCl₄(120 ml) was added benzoylperoxide (25 mg) and the reaction mixturestirred at 85° C. for 10 min. NBS (4.97 g, 28 mmol) and morebenzoylperoxide (25 mg) were added and mixture stirred at reflux for 2hours. Cooling, filtration and evaporation left an oil which waspurified by flash chromatography on silica gel (PE/EtOAc 60:1) to give5.8 g (82% ) 23 as a colorless oil. ¹H-NMR (DMSO-d₆) δ 7.81 (d, J=5.1Hz, 1 H), 7.26 (d, J=5.1 Hz, 1 H), 4.93 (s, 2 H), 1.55 (s, 9 H). MS(m/z) 278 (M+H)⁺.

Synthesis of 24

[0205] To a solution of (±) indoline-2-carboxylic acid (2.33 g, 20 mmol)and Et₃N (5.6 mL, 40 mmol) in MeOH (40 mL) cooled to 0° C. was addedportionwise Boc₂O (5.24 g, 24 mmol). The ice bath was removed and themixture stirred at room temperature for 18 hours. After evaporation ofthe solvent the resulting oil was dissolved in EtOAc and washedsuccessively with hydrochloric acid (1 N) and brine. Drying andevaporation gave 4.48 g (85% ) of a white solid. The N-Boc protectedindoline-2-carboxylic acid (4.48 g, 17 mmol) was dissolved in DMF (50mL) and cesium carbonate (5.54 g, 17 mmol) and benzyl bromide (1.65 mL,16.2 mmol) were added. The resulting solution was stirred at roomtemperature for 24 hours. The reaction mixture was diluted with EtOAcand washed with 1 N aqueous HCl, saturated aqueous NaHCO₃ and brine.Drying and evaporation gave an oil, which was purified by flashchomatography on silica gel (PE/EtOAc 12:1) to give 5.42 g (95% ) ofindoline 24.

Synthesis of 25

[0206] To a solution of KHMDS (0.5 N in toluene, 8 ml, 4 mmol) in THF (6ml) cooled to −78° C. was added dropwise a solution of 24 (706 mg, 2mmol) in THF (4 ml). The resulting solution was stirred at −40° C. for 1hour and then cooled to −78° C., whereupon 23 (1.66 g, 6 mmol) in THF (4ml) was added dropwise. The reaction mixture was allowed to warm to roomtemperature over 5 h, and then diluted with EtOAc (100 ml). The organiclayer was washed with hydrochloric acid (1 N), saturated aqueous NaHCO₃and brine. Drying and evaporation gave an oil, which was purified byflash chromatography on silica gel (PE/EtOAc 8:1) to give 935 mg (85% )of the fully protected alkylated indoline. ¹H-NMR (DMSO-d₆) δ 7.51 (bs,1 H); 7.50 (d, J=5.2 Hz, 1 H), 7.34 (s, 5 H), 7.03 (t, J=8.0 Hz, 1 H),6.91 (d, J=7.3 Hz, 1 H), 6.78 (t, J=7.4 Hz, 1 H), 6.72 (d, J=5.1 Hz, 1H), 5.25 (d, J=12.7 Hz, 1 H), 5.20 (d, J=12.7 Hz, 1 H), 4.16 (d, J=14.2Hz, 1 H), 3.70 (d, J=14.2 Hz, 1 H), 3.29 (s, 2 H), 1.51 (s, 9 H), 1.48(s, 9 H). MS (m/z) 550.7 (M+H)⁺.

[0207] This compound was dissolved in MeOH (50 ml). Pd/C 30% (160 mg)was added and the reaction mixture was stirred at room temperature underhydrogen (atmospheric pressure) for 18 hours. Dilution with EtOAc,filtration and evaporation of the solvent gave 781 mg (100% ) of 25 asan oil. ¹H-NMR (DMSO-d₆) δ 7.52 (bs, 1 H); 7.51 (d, J=5.2 Hz, 1 H), 7.03(t, J=7.4 Hz, 1 H), 6.91 (d, J=7.3 Hz, 1 H), 6.79 (t, J=7.3 Hz, 1 H),6.72 (d, J=5.2 Hz, 1 H), 4.20 (d, J=14.2 Hz, 1 H), 3.70 (d, J=14.2 Hz, 1H), 3.30 (s, 2 H), 1.51 (s, 9 H), 1.49 (s, 9 H).

Example 18 Synthesis of tetrahydroquinoline (27)

[0208]

[0209] A solution of quinaldic acid (5 g, 28.9 mmol) in MeOH (100 mL)was treated with PtO₂ (500 mg) then stirred under a positive pressure ofhydrogen for 4 h. The solution was filtered and concentrated to affordan orange oil which was taken up in DMF (30mL) and treated with cesiumcarbonate (18.4 g, 56.4 mmol) and benzyl bromide (6.71 mL, 56.4 mmol).After stirring for 15 h the mixture was diluted with hydrochloric acid(1 N) and extracted with EtOAc. Concentration of the dried organic layergave 26 as a white solid (5.8 g, 57% ). ¹H NMR (DMSO-d₆) 7.32 (m, 10 H),6.89 (m, 2 H), 6.51 (m, 1 H), 6.39 (d, J=8.1 Hz, 1 H), 5.17 (d, J=7.5Hz, 1 H), 5.13 (d, 1 H, J=7.5 Hz, 1 H), 4.63 (d, J=8.1 Hz, 1 H), 4.40(m, 1 H), 4.39 (d, J=8.1 Hz, 1 H), 2.68 (m, 1 H), 2.55 (m, 1 H), 2.30(m, 1 H), 2.13 (m, 1 H); MS (m/z) 268.3 (MH)⁺

[0210] A solution of 26 (1.50 g, 4.20 mmol) in dry THF (20 mL) wastreated dropwise at −78° C. with LDA (2 M solution in THF, 1.96 mL, 3.92mmol) and the solution was maintained at −78° C. for 1.5 h. A solutionof 23 (3.49 g, 12.6 mmol) in THF (5 mL) was added over 30 min viasyringe pump, and the mixture was warmed to room temperature and stirredfor 15 h. After addition of hydrochloric acid (1 N), the mixture wasextracted with EtOAc. Concentration of the organic layer gave a residuewhich was purified by chromatography (Biotage column, silica gel,40×115, PE/EtOAc 98:2) to give 27 as a solid (994 mg, 43% ). ¹H NMR(DMSO-d₆) 7.63 (d, J=5.2 Hz, 1 H), 7.25 (m, 6 H), 7.15 (m, 2 H), 7.10(m, 2 H), 7.04 (d, J=5.2 Hz, 1 H), 6.84 (m, 2 H), 6.50 (m, 1 H), 6.27(d, J=7.8 Hz, 1 H), 5.12 (m, 2 H), 4.88 (d, J=11.1 Hz, 1 H), 4.49 (d,J=13.7 Hz, 1 H), 3.98 (d, J=10.5 Hz, 1 H), 3.37 (d, J=10.5 Hz, 1 H),2.57 (m, 1 H), 2.43 (m, 1 Hp,2.15 (m, 2 H), 1.42 (s, 9 H)

Synthesis of Inhibitors

[0211] Reactions on solid support were performed either in polyethylenesyringes agitating the reaction mixture on a wheel, or with the Quest210 synthesizer (Argonaut Technologies) using standard solid-phasetechniques.

Example 19 Solid Phase Synthesis of 165

[0212]

Step 1:N2-[(9-Fluorenyl)methoxycarbonyl]-N1-[2-(2,4-dichloropenyl)ethyl]-L-cysteinamide(40)

[0213] A solution of Fmoc-S-trityl-L-cysteine (12.0 g, 20.5 mmol), EDC(4.32 g, 22.55 mmol) and HOBt (3.05 g, 22.55 mmol) was stirred at 0° C.in anhydrous DCM (200 mL) for 15 min. Then 2-(2,4-dichlorophenyl)ethylamine (4.68 g, 24.6 mmol) was added dropwise. The cooling bath wasremoved and the mixture stirred overnight. Most of the DCM was removedin vacuo, the residue was dissolved in EtOAc (300 mL) and subsequentlywashed with aqueous potassium hydrogensulfate (1 M, 2×), water,saturated aqueous sodium hydrogencarbonate and brine. Drying over sodiumsulfate and removal of the solvent gave an off-white foam (16.0 g).

[0214] 14.0 g (18.48 mmol) of this material were stirred in DCMcontaining 15% (TFA) and 5% TES (total volume 200 mL) at roomtemperature. After 10 min the. reaction was evaporated to dryness, thesolid residue was washed three times with 150 mL n-pentane/diethylether(1:1, v/v) and then dried under high vacuum. 8.86 g of 40 compound wasobtained as a colorless solid. ¹H-NMR (DMSO), δ 8.08 (t, J=5.5 Hz, 1 H),7.89 (d, J=7.5 Hz, 2 H), 7.73 (d, J=7.4 Hz, 2 H), 7.54 (s, 1 H), 7.41(t, J=7.4 Hz, 2 H), 7.32 (t, J=7.4 Hz, 2 H), 7.30 (bs, 2 H), 4.32 (m, 1H), 4.27 (m, 2 H), 4.02 (m, 1 H), 3.31 (m, 2 H), 2.82 (t, J=6.9 Hz, 2H), 2.75 (m, 1 H), 2.61 (m, 1 H), 2.22 (t, J=8.4 Hz, 1 H).

Step 2: Synthesis of Resin 41

[0215]1.5 g Trityl chloride resin (Novabiochem, loading 2.05 mmol ofactive chlorine) was washed once with DCM and then added to a solutionof 40 in DCM containing 7% TFA (40 mL). The solution was agitatedovernight (orbital shaker, or slow stirring on a magnetic stirrer). Theresin was isolated on a sintered glass funnel, washed with DCM (5×50mL), then methanol (1×50 mL), again with DCM and dried overnight invacuo. 2.57 g of this material in a 50 mL syringe with a 20 micron fritwere treated with a solution of 20% piperidine in DMF for 1 h. The resinwas drained, washed 3× with DMF and the treatment with 20%piperidine/DMF was repeated. After drainage, the resin was washed withDMF (2×20-25 mL), DMF/DCM(1×25 mL)), DCM (3×20-25 mL), methanol anddiethylether (1×20-25 mL each) and dried in vacuo.

Step 3: Synthesis of Resin 42

[0216] The foregoin resin was acylated with a premixed solution ofFmoc-L-leucine (4.23 g, 12.35 mmol), EDC (2.30 g, 12.35 mmol) and HOBt(1.62 g, 12.35 mmol) in DCM (40 mL) for 3 h. After drainage the resinwas washed with DCM (1×25 mL), DMF (2×20-25 mL), DMF/DCM (1×25 mL), DCM(3×20-25 mL), methanol and diethylether (1×25 mL each). After drying invacuo it was treated with a solution of 20% piperidine/DMF (25 mL, 40min). The resin was drained, washed with DMF (2×20-25 mL), DMF/DCM (1×25mL), DCM (3×20-25 mL), methanol and diethylether (1×20-25 mL each) andthen dried in vacuo.

Step 4:. Synthesis of Resin 43

[0217] 1.6 g of resin 42 were acylated with a premixed solution ofFmoc-(γ-O-tert.-butyl)-L-glutamic acid (3.3 g, 7.7 mmol), EDC (1.48 g,7.7 mmol) and HOBt (1.04 g, 7.7 mmol) in DCM (30 mL) for 5 h. Afterwashing, drying and deprotection with a solution of 20% piperidine/DMF(20 mL, 1 h) as described above in step 3, the resin was drained, washedwith DMF (2×20-25 mL), DMF/DCM (1×25 mL), DCM (3×20-25 mL), methanol anddiethylether (1×20-25 mL each), then dried first under a stream ofnitrogen, and subsequently in vacuo.

Step 5: Synthesis of 165

[0218] To 30 mg of resin 43 (ca. 0.029 mmol of compound) was added DCM(1 mL), followed by diisopropylethylamine (0.2 mL) and isobutylchloroformate (0.038 mL, 0.29 mmol). The resin was agitated for 5 h.Prior to cleavage, the resin was washed with DMF (3×1 mL) and DCM (3×1mL) and then treated with a solution of 50% TFA/45% DCM/5% water (1.5mL) containing TIPS (0.02 mL) for 30 min at room temperature. The resinwas washed with cleavage mixture (1-1.5 mL) and DCM (2 mL). The combinedfiltrates were evaporated, the residue was dissolved in 30% water/70%acetonitrile and lyophilized to obtain crude 165 (21 mg, quantitative)as a white powder. A sample (15 mg) was purified by preparative RP-HPLC(gradient: 60% A, 2 min isocratic, then in10 min to 30% A (RT 11.2 min).Fractions containing the desired product were pooled and lyophilized togive compound 165 (10 mg).

[0219]¹H-NMR (DMSO-d₆) δ 12.0 (bs, 1 H), 7.98 (m, 2 H), 7.93 (d, J=7.7Hz, 1 H), 7.55 (s, 1 H), 7.32 (s, 2 H), 7.27 (d, J=7.7 Hz, 1 H), 4.26(m, 2 H), 3.97 (m, 1 H), 3.72 (d, J=6.6 Hz, 2 H), 3.34 (m, 1 H), 3.27(m, 1 H), 2.81 (t, J=6.9 Hz, 2 H), 2.58-2.72 (m, 2 H), 2.24 (t, J=7.7Hz, 2 H), 2.19 (t, J=8.5 Hz, 1 H), 1.85 (m, 2 H), 1.72 (m, 1 H), 1.58(m, 1 H), 1.45 (m, 2 H), 0.86 (m, 9 H), 0.83 (d, J=6.5 Hz, 3 H). MS(m/z) 635.4, 637.4 (M+H)⁺.

Example 20 Synthesis of Compound 225

[0220]

[0221] To resin 43 (30 mg, see example 19) was added DCM (1 mL), DIEA(0.2 mL), EDC (29 mg, 0.15 mmol) and 1,2-phenylen diacetic acid (56 mg,0.29 mmol). After agitation for 5 h, the resin was drained, washed andcleaved as described in example 19, step 5. Evaporation of the solventsgave the crude product, which was purified by preparative RP-HPLC.Gradient: 60% A, 2 min isocratic, then in 10 min to 30% A (RT 8.3 min).Fractions containing the desired product were pooled and lyophilized togive compound 225 (10 mg, 48% ). ¹H-NMR (DMSO-d₆) δ 12.2 (bs, 2 H), 8.27(d, J=7.6 Hz, 1 H), 7.99 (m, 2 H), 7.95 (d, J=8.1 Hz, 1 H), 7.55 (s, 1H), 7.31 (s, 2 H), 7.22 (m, 1 H), 7.17 (m, 3 H), 4.23 (m, 3 H), 3.68 (d,J=16.1 Hz, 1 H), 3.62 (d, J=16.1 Hz, 1 H), 3.54 (d, J=15.4 Hz, 1 H),3.50 (d, J=15.4 Hz, 1 H), 3.20-3.40 (m, 2 H), 2.81 (t, J=6.9 Hz, 2 H),2.68 (m, 1 H), 2.60 (dd, J=7.6, 14.5 Hz, 1 H), 2.24 (m, 3 H), 1.89 (m, 1H), 1.72 (m, 1 H), 1.56 (m, 1 H), 1.45 (m, 2 H), 0.86 (d, J=6.5 Hz, 3H), 0.82 (d, J=6.5 Hz, 3 H). MS (m/z) 711.3, 713.5 (M+H)⁺.

Example 21 Synthesis of Compound 101

[0222]

Step 1: N2-[(9-Fluorenyl)methoxycarbonyl]-N1[2-phenylethyl]-L-cysteinamide

[0223] The title compound was obtained analogous to procedure describedin to example 19, step 1 from coupling of Fmoc-S-trityl-L-cysteine andphenethylamine and subsequent deprotection with TFA and TES.

Step 2

[0224] Analogous to the procedure described in example 19, step 2,trityl chloride resin (525 mg, Novabiochem, loading 0.4 mmol of activechlorine) was alkylated with the foregoing compound (380 mg, 0.85 mmol)in DCM containing 5% TFA (4 mL). The resin was then deprotected with 20%piperidine/DMF.

Step 3

[0225] Analogous to the procedure described in example 19, step 3, theforegoing resin (450 mg) was acylated with Fmoc-L-leucine (239 mg 0.68mmol) and then treated with 20% piperidine/DMF to cleave the Fmocprotecting group.

Step 4

[0226] Analogous to the procedure described in example 19, step 4, theforegoing resin (270 mg) was acylated withFmoc-(γ-O-tert.-butyl)-L-glutamic acid (77 mg, 0.18 mmol) and thentreated with 20% piperidine/DMF to cleave the Fmoc protecting group.

Step 5

[0227] As described in example 19, step 5, the foregoing resin wasacylated with N-(benzyloxycarbonyloxy)succinimide (90 mg, 0.36 mmol) inDCM (1 mL) and DIEA (0.5 mL). After agitating the mixture overnight theresin was drained and washed as described. After cleavage from theresin, 101 was isolated by preparative RP-HPLC (gradient: 65% A, 3 minisocratic, then in 9 min to 35% A; RT 10.2 min). ¹H-NMR (DMSO-d₆) δ 12.0(bs, 1 H), 7.98 (m, 3 H), 7.45 (d, J=8.1 Hz, 1 H), 7.22-7.38 (m, 7 H),7.18 (m, 3 H), 5.01 (s, 2 H), 4.27 (m, 2 H), 4.01 (m, 1 H), 3.20-3.40(m, 2 H), 2.68 (t, J=7.2 Hz, 2 H), 2.60-2.68 (m, 2 H), 2.25 (t, J=7.6, 2H), 2.16 (t, J=8.4, 1 H), 1.86 (m, 1 H), 1.73 (m, 1 H), 1.59 (m, 1 H),1.46 (m, 2 H), 0.87 (d, J=6.4 Hz, 3 H), 0.83 (d, J=6.3 Hz, 3 H). MS(m/z) 601.4 (M+H)⁺.

Example 22 Synthesis of Compound 102

[0228]

[0229] The resin (160 mg) obtained in example 21 (steps 1 to 3, ca.0.021 mmol of compound) was acylated with FmocValOH (36 mg, 0.105 mmol)analogous to the procedure described in example 19, step 4, and thentreated with 20% piperidine/DMF to cleave the Fmoc protecting group. Theresulting resin was then suspended in DCM (1 mL) and DIEA (0.5 mL).Solid N-(4-carboxymethyl-benzyloxycarbonyloxy)-succinimide (65 mg, 0.21mmol, prepared from methyl-(4-hydroxymethyl)-benzoate andN,N′-disuccinimidyl carbonate according to A. K. Gosh et al, TetrahedronLetters 1992, 33, 2781-2784) was added. After agitating the mixtureovernight the resin was drained and washed with DMF (3×1 mL) and DCM(3×1 mL) and then treated with a solution of 50% TFA/45% DCM/5% water(2.5 mL) containing TIPS (0.02 mL) for 30 min at room temperature. Theresin was washed with cleavage reagent (1-1.5 mL) and DCM (2 mL) and thecombined filtrates were evaporated. The crude methyl ester washydrolyzed with aqueous sodium hydroxide (1 mL, 1 N) in methanol (2 mL)for 20 min at 50° C. After cooling to room temperature, hydrochloricacid (1 mL, 1 N) was added and the mixture diluted withacetonitrile/water (40/60, v/v). Compound 102 was obtained bypreparative RP-HPLC (gradient: 65% A, 3 min isocratic, then in 9 min to35% A; RT 8.2 min; 7 mg, 54% 6). ¹H-NMR (DMSO-d₆) δ 12.7 (bs, 1 H), 7.99(app. d, J=7.3, 2 H), 7.92 (m, 3 H), 7.45 (d, J=7.9 Hz, 2 H), 7.39 (d,J=8.8 Hz, 1 H), 7.26 (m, 1 H), 7.25 (d, J=6.9 Hz, 1 H), 7.18 (d, J=7.2Hz, 2 H), 7.17 (m, 1 H), 5.10 (s, 2 H), 4.28 (m, 2 H), 3.85 (app. t,J=7.6 Hz, 1 H), 3.20-3.30 (m, 2 H), 2.69 (t, J=7.3 Hz, 2 H), 2.60-2.68(m, 2 H), 2.15 (t, J=8.2, 1 H), 1.95 (m, 1 H), 1.60 (m, 1 H), 1.45 (m, 2H), 0.84 (m, 12 H). MS (m/z) 615.5 (M+H)⁺.

Example 23 Synthesis of Compound 247

[0230]

Step 1:N2-[(9-Fluorenyl)methoxycarbonyl]-N1-[2-(2-chloropenyl)ethyl]-L-cysteinamide

[0231] The title compound was obtained analogous to the proceduredescribed in to example 19, step 1 from coupling ofFmoc-S-trityl-L-cysteine (14.0 g, 23.9 mmol) and 2-(2-chlorophenyl)ethylamine (31.07 mmol) and subsequent deprotection with TFA and TES.

Step 2

[0232] Analogous to the procedure described in example 19, step 2,trityl chloride resin (1.8 g, Novabiochem, loading 2.05 mmol/g of activechlorine) was alkylated with the foregoing compound (10.8 g, 22.6 mmol)in DCM containing 5% TFA (45 mL). The Fmoc group was cleaved with 20%pip/DMF.

Step 3

[0233] Analogous to the procedure described in example 19, step 3, theforegoing resin was acylated with a premixed solution of Fmoc-L-leucine(3.70 g, 10.4 mmol), PyBop (5.4 g, 10.4 mmol), DIPEA (20.8 mmol) andHOBt (1.6 g, 10.4 mmol) in DMF (25 mL) overnight. After drainage andwashing the resin was then treated with 20% piperidine/DMF to cleave theFmoc protecting group.

Step 4

[0234] Analogous to the procedure described in example 1, step 4, theforegoing resin was acylated with a premixed solution ofFmoc-(γ-O-tert.-butyl)-L-glutamic acid (4.4 g, 10.4 mmol), PyBop (5.4 g,10.4 mmol), DIPEA (20.8 mmol) and HOBT (1.6 g, 10.4 mmol) in DMF (25 mL)and then treated with 20% piperidine/DMF to cleave the Fmoc protectinggroup.

Step 5

[0235] The foregoing resin (20 mg, 0.022 mmol) in DCM (2 mL) was reactedwith phenylisocyanate (26 mg, 0.22 mmol) and DIPEA (0.046 mL, 0.22 mmol)for 2 h. Then the resin was drained and washed with DMF (2×10 mL), DCM(2×10 mL) and diethyl ether (2×10 mL). Cleavage of the product wasaffected with a mixture of TFA/DCM/water/and TIPS (6:6:1:1; 2 mL) for 10min. This procedure was repeated two times. The combined filtrates wereaged for 40 min, then evaporated. The residue was dissolved in a mixtureof water and acetonitrile (30/70, 3 mL) and lyophilized to give 247 as acolorless powder. ¹H-NMR(DMSO-d₆) δ 8.65 (s, 1H), 8.22 (4,J=7.72, 1H),8.017 (m,2 H), 7.43-7.19 (m, 8 H), 6.89 (t, 7.31 Hz, 1H), 6.41 (d,J=7.84Hz, 1H), 4.33-4.26 (m, 3 H), 3.31 (m,2 H), 2.85 (t,J=7.10 Hz,2 H), 2.71(m,2 H), 2.28-2.16 (m,3 H),1.89-1.45 (m,5 H), 0.89 (d, J=6.61 Hz, 3H),0.85 (d, J=6.61 Hz, 3H); MS (m/z) 619 (M+H)⁺.

Example 24 Synthesis of Compound 252

[0236]

[0237] The resin (20 mg) obtained in example 23, step 4 was acylatedwith 1-naphthalensulfonyl chloride (50 mg, 0.22 mmol) under identicalconditions described in example 23, step 5. After cleavage andlyophilization 252 was obtained as a colorless powder. ¹H-NMR(DMSO-d₆) δ8.65 (d, J=7.96 Hz, 1H), 8.31 (d, J=9.04 Hz, 1 H),8.20 (d, J=8.28, 1 H),8.12-7.92 (m, 5 H), 7.68-7.56 (m, 3 H), 7.40 (m, 1 H), 7.31-7.22 (m, 3H), 4.22 (q, J=7.33 Hz, 1 H), 4.04 (m, 1 H), 3.81 (q, J=5.65, 1H),3.36-3.22 (m, 2 H),2.81 (t, J=5.98 Hz, 2 H)2.67-2.58 (m,2 H),2.14-1.95 (m,3 H),1.76-1.60 (m,2 H),1.28-1.57 (m, 3 H),0.75 (d, J=5.83Hz, 3 H),0.61 (d,J=5.83 Hz, 3 H); MS (m/z) 691 (M+H)⁺.

Example 25 Synthesis of Compound 409

[0238]

Step 1:N2-[(9-Fluorenyl)methoxycarbonyl]-N1-[2-(4-carboxymethyl-phenyl)ethyl]-L-cysteinamide

[0239] The title compound was obtained analogous to the proceduredescribed in to example 19, step 1 from coupling ofFmoc-S-trityl-L-cysteine and amine hydrochloride 5b and subsequentdeprotection with TFA and TES.

Step 2-5

[0240] The procedures are identical to the ones described in example 19,using 20 mg of resin, but in step 3 (2S, 4S)-Fmoc-L-cyclohexyl-alaninewas used instead of Fmoc-Leu-OH. The crude product obtained aftercleavage (22 mg0.033 mmol) was dissolved in methanol (1 mL) and cooledto 0° C. After 5 minutes 1 ml of 1 N NaOH (30 equiv) was added andreaction mixture was left at room temperature. After 90 min solution wasconcentrated in vacuo and ethyl acetate was added. The organic layer waswashed with 1 N HCl (3×), brine and dried. Evaporation of the solventand freeze drying from acetonitrile/water gave 409 (8 mg, 35% ). ¹H-NMR(DMSO-d₆, 300 MHz, 300K) δ 8.00-7.92 (m, 3 H), 7.85 (d, J=8.19 Hz, 2 H),7.33 (d, J=8.19, 2 H), 7.28 (m, 1 H), 4.36-4.22 (m, 2 H), 3.97 (m, 1 H),3.73 (d, J=6.63 Hz, 2 H), 2.79 (t, J=6.92, 2 H), 2.68-2.62 (m, 2 H),2,2-2,1 (m, 3 H), 1.9-1.0 (m, 18 H), 0.877 (d, J=6.63 Hz, 6 H). MS (m/z)651.5 (M+H)⁺.

Example 26 Synthesis of Compound 506

[0241]

[0242] The procedure is identical to the one described in example 23,but in step 3 (2S,4S)-Fmoc-4-phenyl-pyrrolidine-2-carboxylic acid wasused instead of Fmoc-Leu-OH and in step 5 isobutyl chloroformate wasused for the acylation. ¹H-NMR(DMSO-d₆) δ 8.13 (d, J=8.08 Hz, 1 H),7.99(t, J=3.8 Hz, 1H), 7.43-7.20 (m, 10H), 4.54 (t, J=4.96 Hz, 1H), 4.33 (q,J=7.89 Hz,2 H), 4.15 (m, 1 H),3.71 (d, J=6.52 Hz, 2 H),3.55( m,2 H),3.39-3.28 (m,2 H),2.86 (t, J=7.11 Hz, 2 H), 2.78-2.65 (m, 2 H),2.48-2.23 (m, 5H),1.94-1.24 (m,3H), 0.86 (d, J=6.6 Hz, 6H); MS (m/z) 661(M+H)⁺.

Example 27 Synthesis of Compound 177 and 178 Step 1: Synthesis of Resin44

[0243] A solution of Fmoc-S-trityl-L-cysteine (10.0 g, 17.1 mmol), inDCM containing 15% trifluoroacetic acid (TFA) and 5% TES (total volume150 mL) was stirred at room temperature. After 10 min the reaction wasevaporated to dryness, the solid residue was washed three times with 150mL n-pentane and then dried under high vacuum. 4.46 g of Fmoc-L-cysteinewas obtained as a colorless solid. This material was dissolved in DCM(20 mL) containing 7% TFA. 1.0 g Trityl chloride resin (Novabiochem,loading 2.05 mmol of active chlorine) was added in one portion. Thesolution was slowly agitated overnight (orbital shaker, or slow stirringon a magnetic stirrer). The resin was isolated on a sintered glassfunnel, washed with DCM (5×40 mL), methanol (1×40 mL) and again with DCMand dried overnight in vacuo.

Step 2: Synthesis of 45

[0244] To 40 mg (loading ca. 1.25 mmol/g, 0.05 mmol) of resin 44 wasadded a solution of 7a (53 mg, 0.26 mmol), EDC (50 mg, 0.26 mmol) andHOBt (40 mg, 02.6 mmol) in DCM (1.5 mL), followed by DIEA (0.2 mL). Thereaction was agitated overnight, the resin was washed with DMF (2×3 mL),DCM/DMF (1×3 mL), DCM (1×), methanol (1×) and DCM (3×3 mL). Deprotectionof the Fmoc-group was achieved by treatment with 20% piperidine/DMF (3mL) for 1 h, then washing the resin with DMF (2×3 mL) and againtreatment with 20% piperidine/DMF. After drainage, the resin was washedwith DMF (2×3 mL), DCM/DMF (1×3 mL), DCM (1×), methanol (1×) and DCM(3×3 mL) and dried under a stream of nitrogen.

Step 3: Synthesis of Resin 46

[0245] To resin 45 was added a solution of Fmoc-L-leucine (92 mg, 0.26mmol), EDC (50 mg, 0.26 mmol) and-HOBt (40 mg, 0.26 mmol) in DCM(1.5mL). After 4 h the resin was washed as described in step 2, then treatedwith 3 mL of 20% piperidine/DMF for 1 h. The resin was washed asdescribed in step 2, and dried under a stream of nitrogen.

Step 4: Synthesis of Resin 47

[0246] Compound 22 (79,mg, 0.26 mmol), EDC (50 mg) and HOt (40 mg) inDCM (1.5 mL) was added to resin 46 and agitated for 4 h. The resin waswashed and dried as described in step 3.

Step 5: Synthesis of 177

[0247] Resin 47 was treated with a solution of 50% TFA/45% DCM/5% water(2.5 mL) containing TES (0.02 mL) for 30 min at room temperature. Afterdrainage, the resin was washed with cleavage reagent (2 mL) and DCM (3mL) and the combined filtrates were evaporated. The residue wasdissolved in 30% water/70% acetonitrile and lyophilized to obtain methylester 177 (30 mg, ca.91% crude) as a white powder. A sample (7 mg) ofthe compound was purified by preparative RP-HPLC (gradient: 65% A, 3 minisocratic, then in 9 min to 35% A; RT 10.7 min; 4 mg). ¹H-NMR (DMSO-d₆)δ 8.0 (m, 2 H), 7.92 (d, J=7.7 Hz, 1 H), 7.89 (d, J=1.6 Hz, 1 H), 7.81(dd, J=1.6, 7.95 Hz, 1 H), 7.45 (d, J=7.9 Hz, 1 H), 7.25 (d, J=7.6 Hz, 1H), 4.25 (m, 2 H), 3.95 (app. dd, J=8.2, 13.7 Hz, 1 H), 3.84 (s, 3 H),3.71 (d, J=6.6 Hz, 2 H), 3.37 (m, 2 H), 2.90 (t, J=6.8 Hz, 2 H), 2.69(m, 1 H), 2.62 (m, 1 H), 2.23 (t, J=7.6 Hz, 2 H), 2.19 (t, J=8.6 Hz, 1H), 1.82 (m, 2 H), 1.71 (m, 1 H), 1.58 (m, 1 H), 1.44 (m, 2 H), 0.86 (m,9 H), 0.82 (d, J=6.5 Hz, 3 H). MS (m/z) 659.5, 661.0 (M+H)⁺.

Step 6: Synthesis of 178

[0248] The remaining methyl easter 177 (23 mg) was dissolved in methanoland THF (1 mL each) and 1 N sodium hydroxide (1 mL) was added dropwise.After 15 min 1 N HCl (1 mL) was added and the solution diluted withwater/acetonitrile (50/50, v/v). Compound 178 was isolated bypreparative RP-HPLC (gradient: 65% A, 3 min isocratic, then in 9 min to35% A; RT 7.2 min; 13 mg). ¹H-NMR (DMSO-d₆) δ 8.0 (m, 2 H), 7.93 (d,J=7.7 Hz, 1 H), 7.87 (d, J=1.5 Hz, 1 H), 7.79 (dd, J=1.5, 7.9 Hz, 1 H),7.42 (d, J=7.9 Hz, 1 H), 7.25 (d, J=7.8 Hz, 1 H), 4.25 (m, 2 H), 3.96(app. dd, J=8.1, 13.7 Hz, 1 H), 3.71 (d, J=6.6 Hz, 2 H), 3.37 (m, 2 H),2.90 (t, J=6.9 Hz, 2 H), 2.70 (m, 1 H), 2.62 (m, 1 H), 2.23 (t, J=7.7Hz, 2 H), 2.19 (t, J=8.3 Hz, 1 H), 1.83 (m, 2 H), 1.72 (m, 1 H), 1.58(m, 1 H), 1.44 (m, 2 H), 0.86 (m, 9 H), 0.82 (d, J=6.5 Hz, 3 H). MS(m/z) 645.5, 647.5 (M+H)⁺.

Example 28 Synthesis of Compound 181

[0249]

[0250] Resin 46 (200 mg) (obtained as in example 27, steps 1 to 3) wasacylated with a premixed solution of Fmoc-L-Valine (441 mg, 1.3 mmol),EDC (249 mg, 1.3 mmol) and HOBt (199 mg, 1.3 mmol) for 5 h. Afterwashing and drying of the resin as described in example 27, step 2, theFmoc group was deprotected with 20% piperidine/DMF (6 mL) for 1 h. Afterdrainage and washing (step 2, example 27) the resin was dried under astream of nitrogen.

[0251] The acylating reagentcyclopentylmethyloxycarbonyl-N-hydroxysuccinimide was prepared in situby addition of cyclopentanol (50 mg, 0.5 mmol) and triethylamine (152mg, 1.5 mmol) to a solution of N,N′-disuccinimidyl carbonate (128 mg,0.5 mmol) in anhydrous acetonitrile (2 mL) (see A. K. Gosh et al,Tetrahedron Letters 1992, 33, 2781-2784). After 30 min stirring at roomtemperature the solution was diluted with DCM (2 mL) and added to 40 mg(0.05 mmol) of the foregoing resin. After agitation for 5 h, the resinwas drained and washed as described above. It was then treated with asolution of 50% TFA/45% DCM/5% water (2.5 mL) containing TES (0.02 mL)for 30 min at room temperature. After drainage, the resin was washedwith cleavage reagent (2 mL) and DCM (3 mL). The combined filtrates wereevaporated, the residue was dissolved in 30% water/70% acetonitrile andlyophilized to obtain the methyl ester of 181 as a white powder. Thispowder was dissolved in methanol (1 mL) and 1 N sodium hydroxide (1 mL)was added dropwise. After 30 min 1 N HCl (1 mL) was added and thesolution diluted with water/acetonitrile (60/40, v/v). Compound 181 waspurified by preparative RP-HPLC (gradient: 65% A, 5 min isocratic, thenin 7 min to 30% A; RT 12.8 min; 8 mg, 25% ). ¹H-NMR (DMSO-d₆) δ 8.06(bs, 1 H), 7.96 (m, 2 H), 7.88 (d, J=1.5 Hz, 1 H), 7.79 (dd, J=1.5, 7.9Hz, 1 H), 7.44 (d, J=7.9 Hz, 1 H), 7.10 (d, J=8.6 Hz, 1 H), 4.26 (m, 2H), 3.83 (d, J=7.0 Hz, 2 H), 3.82 (m, 1 H), 3.35 (m, 2 H), 2.91 (t,J=6.7 Hz, 2 H), 2.55-2.70 (m, 2 H), 2.21 (t, J=8.6 Hz, 1 H), 2.11 (m, 1H), 1.94 (m, 1 H), 1.33-1.78 (m, 9 H), 1.21 (m, 2 H), 0.88 (d, J=6.5 Hz,3 H), 0.83 (m, 9 H). MS (m/z) 661.7, 643.7 (M+H)⁺.

Example 29 Synthesis of Compounds 135 and 134

[0252]

[0253] The synthesis of compound 135 proceeds analogous to theprocedures described in example 27, steps 2 to 5, using 60 mg of resin44 and 6 (100 mg, 0.39 mmol). After cleavage from the resin, 135 wasisolated by preparative reversed-phase HPLC. Gradient: 65% A, 5 minisocratic, then in 7 min to 35% A. Fractions containing the desiredproduct (RT 12.4 min) were pooled and lyophilized. ¹H-NMR (DMSO-d₆) δ7.96 (m, 2 H), 7.92 (d, J=7.8 Hz, 1 H), 7.61 (d, J=16.1 Hz, 1 H), 7.55(s, 1 H), 7.53 (m, 1 H), 7.32 (t J=7.6 Hz, 1 H), 7.25 (m, 2 H), 6.60 (d,J=16.1 Hz, 1 H), 4.25 (m, 2 H), 4.17 (q, J=7.1 Hz, 2 H), 3.96 (m, 1 H),3.71 (d, J=6.6 Hz, 2 H), 3.23-3.50 (m, 2 H), 2.73 (t, J=7.2 Hz, 2 H),2.66 (m, 1 H), 2.2.61 (m, 1 H), 2.24 (t, J=7.6 Hz, 2 H), 2.13 (t, J=8.2Hz, 1 H), 1.83 (m, 2 H), 1.76 (m, 1 H), 1.59 (m, 1 H), 1.45 (m, 2 H),1.25 (t, J=7.1 Hz, 3 H), 0.86 (m, 9 H), 0.82 (d, J=6.5 Hz, 3 H). MS(m/z) 665.5 (M+H)⁺.

Synthesis of 134

[0254] Crude 135 from a similar experiment was dissolved in methanol (1mL) and 1 N sodium hydroxide (1 mL) was added dropwise. After 15 min 1 NHCl (1 mL) was added and the solution diluted with water/acetonitrile(50/50, v/v). The compound was purified by preparative RP-HPLC with asdescribed for 134. Fractions containing 134 (RT 9.1 min) were pooled andlyophilized. ¹H-NMR (DMSO-d₆) δ 7.97 (m, 2 H), 7.92 (d, J=7.8 Hz, 1 H),7.55 (d, J=16.0 Hz, 1 H), 7.51 (s, 1 H), 7.50 (m, 1 H), 7.31 (t J=7.5Hz, 1 H), 7.25 (m, 2 H), 6.50 (d, J=16.0 Hz, 1 H), 4.27 (m, 2 H), 3.97(app. dd, J=8.0, 13.5 Hz, 1 H), 3.72 (d, J=6.6 Hz, 2 H), 3.40-3.50 (m, 2H), 2.73 (t, J=7.1 Hz, 2 H), 2.60-2.68 (m, 2 H), 2.24 (t, J=7.6 Hz, 2H), 2.13 (t, J=8.2 Hz, 1 H), 1.83 (m, 2 H), 1.76 (m, 1 H), 1.58 (m, 1H), 1.44 Em, 2 H), 0.86 (m, 9 H), 0.82 (d, J=6.5 Hz, 3 H). MS (m/z)637.5 (M+H)⁺.

Example 30 Synthesis of Compound 109

[0255]

[0256] The synthesis of compound 109 proceeds analogous to theprocedures described in example 27, steps 2 to 5, using 40 mg of resin44 and 2 (38 mg, 0.26 mmol). After cleavage from the resin, 109 (15 mg,50% ) was isolated by preparative reversed-phase HPLC. Gradient: 65% A,5 min isocratic, then in 7 min to 30% A; RT 11.8 min. ¹H NMR: (300 MHz,DMSO-d₆): δ 7.98 (d, J=8.1 Hz, 1 H); 7.89 (m, 2 H); 7.24(m, 5 H); 7.1(m, 1 H); 4.29 (m, 2 H); 3.96 (m, 1 H); 3.73 (d, J=6.6 Hz, 2 H); 3.47(dd, J, =13.8 Hz, J₂=6.2 Hz, 1 H); 3.23 (dd, J₁=13.7 Hz, J₂=5.3 Hz, 1H); 2.60 (m, 2 H); 2.24 (t, J=7.8 Hz, 2 H); 2.09 (t, J=8.1 Hz, 1 H);1.84 (m, 2 H); 1.73 (m, 1 H); 1.60 (m, 1 H); 1.43 (m, 2 H); 0.85((m, 14H); 0.74 (m, 2 H). MS (m/z) 593 (M+1)⁺.

Example 31 Synthesis of Compound 188

[0257]

[0258] The synthesis of compound 188 proceeds analogous to theprocedures described in example 27, steps 2 to 5, using 40 mg of resin44 and 3 (71 mg, 0.26 mmol). After cleavage from thee resin, 188 (20 mg,58% ) was isolated by preparative reversed-phase HPLC. Gradient: 65% A,5 min isocratic, then in 7 min to 30% A; RT 9.5 min). ¹H NMR (300 MHz,DMSO-d₆): δ 8.04 (m, 2 H) 7.96 (d, J=7.8 Hz, 1 H); 7.55 (d, J=7.8 Hz, 1H); 7.33 (d, J=8.1 Hz, 1 H); 7.28 (d, J=8.0 Hz, 1 H); 7.14 (s, 1 H);7.12 (t, J=7.1 Hz, 1 H); 7.03 (t, J=6.9 Hz, 1 H); 4.93 (s, 2 H); 4.31(m, 2 H); 3.99 (dd, J₁=8.0 Hz, J₂=5.5 Hz, 1 H); 3.73 (d, J=6.6 Hz, 2 H);3.32 (m, 2 H); 2.81 (t, J=7.7 Hz, 2 H); 2.71 (m, 2 H); 2.24 (m, 3 H);1.85 (m, 2 H); 1.74 (m, 1 H); 1.60 (m, 1 H); 1.49 (m, 2 H); 0.88 (n, 12H). MS (m/z) 664 (M+1)⁺.

Example 32 Synthesis of Compound 190

[0259]

[0260] The synthesis of compound 190 proceeds analogous to theprocedures described in example 27, steps 2 to 5, using 40 mg of resin44 and 1 (33 mg, 0.26 mmol). After cleavage from the resin, 190 wasisolated by preparative reversed-phase HPLC. Gradient: 65% A, 5 minisocratic, then in 7 min to 30% A; RT 10.3 min. ¹H NMR (300 MHz,DMSO-d₆): δ 12.0 (bs, 1 H), 7.98 (m, 2 H) 7.93 (d, J=7.8 Hz, 1 H), 7.43(dd, J=3.0, 4.9 Hz, 1 H),7.26 (d, J=7.7 Hz, 1 H), 7.18 (bs, 1 H); 6.99(d, J=4.9, 1 H); 4.27 (m, 2 H); 3.97 (app dd, J=7.6, 13.4 Hz, 1 H); 3.72(d, J=6.6 Hz, 2 H); 3.34 (m, 2 H); 2.72 (t, J=7.1 Hz, 2 H); 2.59-2.80(m, 2 H); 2.24 (t, J=7.8 Hz, 2 H);. 2.16 (t, J=8.1 Hz, 1 H); 1.83 (m, 2H); 1.72 (m, 1 H); 1.59 (m, 1 H); 1.45 (m, 2 H); 0.86 (m, 9 H), 0.83 (d,J=6.6 Hz, 3 H). MS (m/z) 573 (M+1)⁺.

Example 33 Synthesis of Compound 163

[0261]

Synthesis of Resin 48

[0262] The synthesis of resin 48 is analogous to the proceduresdescribed in example 27 steps 2 to 4, using 120 mg of resin 44 and(4-(2-aminoethyl)-sulfonamide (156 mg, 0.78 mmol). For this couplingstep a mixture of DMF/DCM(1/1, v/v, 3 mL) was used. Fmoc-L-Leucine and22 were coupled as described to give resin 48.

Acylation of Resin 48

[0263] 30 mg of resin 48 (0.038 mmoles) were suspended in DCM (2 mL) andtreated with EDC (73 mg, 0.38 mmoles), DMAP (46 mg, 0.38 mmoles), andfusaric acid (68 mg, 0.38 mmoles) for 4 h. The resin was drained, washedwith DMF and DCM, and the coupling repeated. Then the resin was drained,washed (2×DMF, 2×DCM) and dried under vacuum. Cleavage was achieved bytreating the resin with TFA/DCM/H₂O 50:45:5 (2 mL) in the presence of ofTES (0.02 mL) for 30 minutes, followed by filtration. Evaporation of thefiltrate afforded 35 mg of an orange oil which was purified bypreparative RP-HPLC. Gradient: 70% A, 5 min isocratic, then in 7 min to30% A. Compound 163 was obtained as the trifluoroacetate afterlyophilization. ¹H NMR (300 MHz, DMSO-d₆): δ 8.55 (s, 1 H); 7.99 (m, 2H); 7.94(d, J=8.3 Hz, 2 H); 7.94 (m, 1 H); 7.88 (s, 1 H); 7.87 (d, J=1.8Hz, 1 H); 7.46(d, J=8.3 Hz, 2 H); 7.28(d, J=7.8 Hz, 1 H); 4.25 (m, 2 H);3.97 (m, 1 H); 3.73 (d, J=6.7 Hz, 2 H); 2.81 (t, J=6.9 Hz, 2 H); 2.68(m, 4 H); 2.25 (d, J=6.7 Hz, 2 H); 2.15 (t, J=8.0 Hz, 1 H); 1.80 (m, 3H); 1.57 (m, 3 H); 1.46 (m, 2 H); 1.29 (m, 2 H); 0.86 (m, 15 H). MS(m/z) 807 (M+1)⁺.

Example 34 Synthesis of Compound 321

[0264]

[0265] For the synthesis of 321 the protected pentapeptideAc-Asp(OtBu)-Glu(OtBu)-Dif-Glu(OtBu)-ChaOH (49) was used. Peptide 49 canbe synthesized by classical solid-phase synthetic methods, or by asolution phase protocol using Fmoc-protection and deprotection of theFmoc group with 4-AMP.

Step 1: Synthesis of 50

[0266] Acid 14c (700 mg, 1.94 mmol) was coupled to amine hydrochloride 5(549 mg, 2.13 mmol) in DCM (20 mL) using EDC (409 mg, 2.13 mmol) andHOBt (326 mg, 2.13 mmol) in the presence of DIPEA (376 mg, 2.91 mmol)overnight at room temperature. An acid-base extractive workup gave thecrude product, which was purified by flash chromatography (PE/EtOAC 3:1,1% ethanol) to give a colorless foam (787 mg). 230 mg (0.42 mmol) ofthis material were dissolved in DCM (8 mL) and treated with 4-AMP (1.92g, 16.8 mmol). After 20 min EtOAc (60 mL) was added and the solutionthoroughly extracted with phosphate buffer (pH 5.5). After dryingiandevaporation amine 50 (135 mg, 94% ) was obtained as a colorless oil insufficient purity. ¹H-NMR (DMSO-d₆) δ 8.07 (bs, 1 H); 7.81 (d, J=7.9 Hz,1 H); 7.32 (d, J=7.9 Hz, 1 H); 6.08 (tt, J=56.9, 4.5 Hz, 1 H); 3.3-3.4(m, 2 H); 2.79 (t, J=6.9 Hz, 2 H); 2.0-2.2 (m, 1 H); 1.8-1.95 (m, 1 H);1.53 (s, 9 H). MS (m/z) 343 (M+H)⁺.

Step 2: Synthesis of 321

[0267] Pentapeptide 49 (50 mg, 0.051 mmol) was dissolved in DMF (0.8mL). HATU (21 mg, 0.056 mmol) and amine 50 (20 mg, 0.06 mmol) wereadded, followed by 2,6-lutidine (11 mg, 0.10 mmol). After stirringovernight, EtOAc (50 mL) was added, the solution was extracted withhydrochloric acid (1 N), saturated aqueous sodium hydrogen carbonate andbrine. Drying and evaporation gave a colorless solid, which wasdissolved in a mixture of DCM/TFA/water (60:35:5, 10 mL) and left for 30min. Evaporation gave an offwhite solid, which was purified bypreparative RP-HPLC. Gradient: 70% A, 3 min isocratic, then in 7 min to35% A; RT 8.6 min. ¹H-NMR (DMSO-d₆) δ 8.15 (d, J=7.5 Hz, 1 H); 8.08 (d,J=8.1 Hz, 1 H); 7.80-8.00 (m, 6 H); 7.41 (d, J=6.6 Hz, 1 H); 7.02-7.38(m, 14 H); 5.96 (tt, J=56.1, 4.6 Hz, 1 H); 5.19 (app. t, J=9.3 Hz, 1 H);4.48 (m, 1 H); 4.38 (d, J=10.4 Hz, 1 H); 4.34 (m, 1 H); 4.01-4.18 (m, 3H); 3.4 (m, 2 H, under water from DMSO); 2.78 (t, J=7.0 Hz, 2 H);2.39-2.52 (m, 2 H); 1.98-2.20 (m, 6 H); 1.84 (s, 3 H); 1.50-1.85 (m, 9H); 1.33-1.40 (m, 2 H); 1.08-1.31 (m, 4 H); 0.74-0.92 (m, 2 H). MS (m/z)1079 (M+H)⁺.

Example 35 Synthesis of Compound 522

[0268]

Step 1: Synthesis of 51

[0269] To a stirred solution of Fmoc-S-trityl-cysteine (321 mg, 0.548mmol) in DCM (3 mL) were 7b (140 mg, 0.548 mmol), HOBt (89 mg, 0.659mmol) and EDC (126 mg, 0.659 mmol). The reaction mixture was stirred atroom temperature for 8 hours, diluted with DCM and extracted with HCl (1N), NaOH (1 N), brine. The oily residue was dissolved in CHCl₃ (5 mL)and the solution was cooled in an ice bath. 1.25 mL of 4-AMP were addedand the mixture was stirred at room temperature for 1 h. The solvent wasevaporated under vacuo and the residue was purified by flash columnchromatography on silica gel, eluting with PE/EtOAc (80/20 v/v and then20/80 v/v) to give 51 (330 mg, 60% ). ¹H NMR (DMSO-d₆) δ 7.92 (t, J=6.0Hz, 1 H), 7.81 (d, J=1.6 Hz, 1 H), 7.65 (dd, J=8.0, 1.6 Hz, 1 H),7.38-7.22 (m, 16 H), 3.30-3.20 (m, 2 H), 3.06 (dd, J=7.6, 5.6 Hz, 1 H),2.88 (t, J=6.6 Hz, 2 H), 2.33-2.29 (m, 1 H), 2.12 (dd, J=7.79, 11.6 Hz,1 H), 1.53 (s, 9 H).

Step 2: Synthesis of 52

[0270] To a solution of 51 (190 mg, 0.316 mmol) and(2S,4S)-Fmoc-4-phenyl-pyrrolidine-2-carboxylic acid (131 mg, 0.316 mmol)in CH₂Cl₂ (2 mL), HOBt (51 mg,0.379 mmol) and EDC (73 mg,0.379 mmol)were added and the resulting mixture was stirred at room temperatureover night. After a work-up as described above and deprotection of theFmoc group as described in step 1, the residue was purified by flashcolumn chromatography, eluting with petroleum ether/ethyl acetate (80/20and then 20/20 v/v) to obtain 52 as a white solid in (130 mg, 53% ). MS(m/z) 774.4 (M+1)⁺.

Step 3: Synthesis of 522

[0271] To a solution of 52 (40 mg, 0.052 mmol) in CHCl₃ (2 mL) wereadded Fmoc-cyclopentylglycine (28 mg, 0.077 mmol), HOBt (14 mg, 0.103mmol) and EDC (20 mg, 0.103 mmol) 0.014 g (0.103 mmol). After beingstirred at room temperature for 24 hours, the mixture was diluted withDCM and extracted as described in step 1. The organic layer was dried(Na₂SO₄) and concentrated in vacuo. The oily residue was purified byflash column chromatography on silica gel eluting with PE/EtOAc (1/1v/v) to yield the coupling product (30 mg), which was deprotected with4-AMP (30 equ.) in CHCl₃ (1 mL) at 0° C. for 0.5 hr. After evaporationof the solvent, the crude was filtered on silica gel eluting withPE/EtOAc (4:1) to remove fulvene, then PE/EtOAc (1:1.7) to obtain theamine. This compound was dissolved in DCM (1 mL) and 0.1 mL of DIPEA andthe solution was cooled at 0° C. and treated with 1 equiv. ofisobutylchloroformate. The reaction mixture was allowed to warm up toroom temperature, diluted with DCM and partitioned with HCl (1 N). Theorganic extract was washed with brine, dried over Na₂SO₄, filtered andconcentrated under vacuum. The oily residue was stirred for 4 hours in amixture of DCM/TFA (1:1, 1 mL) in the presence of 20 equ. of TES. Afterevaporation of the solvents, the residue was purified by preparativeRP-HPLC (RP 18, 7 um) Gradient: 70% A to 100% of B in 15 min. 8 mg(0.011 mol) of 522 were isolated. ¹H NMR (DMSO-d₆, 300 MHz, 300 K) δ8.07 (d, J=8.2. Hz, 1 H), 7.99 (t, J=5.4 Hz, 1 H), 7.88 (s, 1 H), 7.80(dd, J=7.9 Hz, J=1.4 Hz, 1 H), 7.45 (d, J=7.9 Hz, 1 H), 7.35-7.29 (m, 5H), 4.52 (dd, J=7.4 Hz, J=2.5 Hz, 1 H), 4.28 (m, 1 H), 4.42-4.05 (m, 2H), 3.73-3.58 (m, 4 H), 3.41 (m, 1 H), 2.92 (t, J=6.7 Hz, 2 H),2.80-2.67 (m, 2 H), 2.35 (t, J=8.7 Hz, 1 H), 2.30-2.16 (m, 3 H),1.82-1.15 (m, 10 H) 0.92-0.84 (m, 6 H).

Example 36 Synthesis of Compound 523

[0272]

Step 1: Synthesis of 53

[0273] EDC (0.9 g, 4.8 mmol) and HOBt (0.7 g, 4.8 mmol) were added to astirred solution of compound 14a (1.2 g, 4.4 mmol) in DCM (15 ml). Themixture was cooled at 0° C. in an ice bath, then a solution of compound8 (1.2 g, 4.4 mmol), DIPEA (0.8 ml, 4.4 mmol) in DCM (5 ml) was addeddropwise over 5 min. The ice bath was removed and the reaction stirredat room temperature for 4 h(TLC: PE/EtOAc 2/1+1% acetic acid). Thereaction was diluted with EtOAc (200 ml) and washed with 1 M HCl (2×50ml), saturated aqueous sodium hydrogencarbonate and brine. The crudeproduct was purified by flash chromatography (PE/EtOAc 3:1 +l% EtOH) togive 1.73 g of coupling product, which was immediately deprotected.

[0274] The foregoing compound was dissolved in MeOH (25 ml) and cooledto 0° C., 100 mg of Pd/C (10% Pd) were added, the ice bath removed andthe reaction was stirred under hydrogen atmosphere for 1 h. Afterremoval of the catalyst by filtration and evaporation of the solvent,the residue was dissolved in DCM and hydrogenchloride (3.6 ml, 1 Msolution in diethyl ether). After removal of the solvents 1.42 g (78% )of amine hydrochloride 53 were obtained as a colorless solid. ¹H-NMR(CDCl₃) δ 1.52 (s, 9 H), 2.53 (m, 2 H), 2.89 (m, 2 H), 3.26 (m, 1 H),3.59 (m, 1 H), 4.52 (bs, 1 H), 6.04 (bt, J=55.2 Hz, 1 H), 7.39 (d,J=7.5, Hz 2 H), 8.16 (s, 1 H), 8.32 (s, 3 H). MS (m/z) 379 ((M+H)⁺, freeamine).

Step 2: Synthesis of 54

[0275] (2S,4S)-Fmoc-4-phenyl-pyrrolidine-2-carboxylic acid (269 mg, 0.65mmol) was dissolved in DCM (5 mL), EDC (138 mg, 0.72 mmol) and HOBt (110mg, 0.72 mmol) were added at 0° C., followed by 52 (270 mg, 0.65 mmol)and DIPEA (126 mg, 0.98 mmol). The ice-bath was removed and the solutionstirred overnight. Another 5 mL of DCM were the added, together with4-AMP (2.97 g, 26 mmol). After 30 min the reaction was diluted withEtOAc (100 mL) and extracted thoroughly with phosphate buffer (pH 5.5).54 was obtained as a yellow foam (346 mg, 96% ), which was used withoutfurther purification. ¹H NMR (300 MHz, DMSO-d₆): δ 8.43 (d, J=8.4 Hz, 1H) 8.25 (t, J=5.8 Hz, 1 H); 7.43 (d, J=7.3 Hz, 2 H); 7.13-7.30 (m, 5 H),5.95 (app tt J=4.5, 56.1 Hz, 1 H), 4.38 (m, 1 H), 3.90 (bd, J=5.9 Hz, 1H), 3.18-3.39 (m, 4 H), 3.15 (app. t, J=8.4 Hz, 1 H); 2.91 (t, J=9.5 Hz,1 H); 2.79 (t, J=6.6 Hz, 2 H); 2.0-2.26 (m, 4 H); 1.49 (s, 9 H). MS(m/z) 552.2 (M+1)⁺.

Step 3: Synthesis of 523

[0276] To a solution of 21 (28 mg, 0.13 mmol) in DMF (1 mL) was addedHATU (49 mg, 0.13 mmol), 2,6-lutidine (28 mg, 0.26 mmol) and solid 54(69 mg, 0.12 mmol). The reaction was stirred for 3 h at ambienttemperature, then taken into EtOAc and extracted with hydrochloric acid(1 N, 2×), water, saturated aqueous sodium hydrogen carbonate (2×) andbrine. The crude product obtained was deprotected with a mixture ofTFA/DCM/water (60:35:5, 10 mL) for 30 min at room temperature.Evaporation gave an oil, which was purified by preparative RP-HPLC(gradient: 60% A, 3 min isocratic, then in 9 min to 35% A; RT 12.3 min;51 mg, 62% ). ¹H-NMR (400 MHz, DMSO-d₆) δ 8.17 (d, J=8.6 Hz, 1 H), 7.99(t, J=5.6 Hz, 1 H), 7.50 (app. d, J=7.5 Hz, 2 H), 7.19-7.35 (m, 5 H),7.03 (bs, 1 H), 6.02 (ddt, J=3.1, 5.9, 54.4 Hz, 1 H), 4.47 (bs, 1 H),4.37 (m, 1 H), 4.08 (t, J=6.3 Hz, 2 H), 3.73 (d, J=6.6 Hz, 2 H), 3.61(m, 2 H), 3.42 (m, 1 H), 3.30 (m, 1 H), 2.85 (t, J=6.8 Hz, 2 H),2.05-2.31 (m, 4 H); 2.05 (m, 1 H); 1.81 (m, 1 H), 0.88 (m, 12 H). MS(m/z) 695.2 (M+H)⁺.

Example 37 Synthesis of Compound 526

[0277]

Synthesis of 55

[0278] Coupling of 53 (54 mg, 0.13 mmol) andN-Fmoc-O-benzyl-L-trans-4-hydroxyproline (53 mg, 0.12 mmol) was carriedout in DCM (2 mL) using HATU (69 mg, 0.18 mmol) and DIPEA (0.042 mL,0.24 mmol) as described in example 36, step 2.

[0279] Deprotection with 4-AMP, gave 55 (65 mg, 90% ) as a colorlessoil. MS (m/z) 582.4 (M+H)⁺.

Synthesis of 526

[0280] Using the procedure described in example 36, step 3, 21 (26 mg,0.12 mmol 4) was coupled to 55 (65 mg, 0.11 mmol) in DCM (2 mL). Afterworkup and deprotection the crude product was purified by preparativeRP-HPLC (Symmetry C18, 19×300 mm; flow 15 mL/min; gradient: 70% A, 3 minisocratic, then in 10 min to 30% A, 2 min isocratic, in 1 min to 10% A;RT 16.2 min; 30 mg (38% ). ¹H-NMR (400 MHz, DMSO-d₆) δ 8.37 (d, J=8.4Hz, 1 H), 8.14 (t, J=5.7 Hz, 1 H), 7.50 (app. d, J=7.2 Hz, 2 H),7.22-7.40 (m, 6 H), 6.06 (bt, J=56.6 Hz, 1 H), 4.52 (d, J=11.5 Hz, 1 H),4.45 (d, J=11.5 Hz, 1 H), 4.30 (m, 2 H), 4.24 (bs, 1 H), 4.12 (d, J=11.1Hz, 1 H), 3.96 (bd, J=8.5 Hz, 1 H), 3.62-3.75 (m, 3 H), 3.37 (m, 1 H),3.24 (m, 1 H), 2.83 (t, J=6.7 Hz, 2 H), 2.20 (m, 1 H), 2.14 (m, 2 H);1.93 (m, 2 H); 1.80 (m, 1 H), 0.86 (m, 12 H). MS (m/z) 725.5 (M+H)⁺.

Example 38 Synthesis of Compound 539

[0281]

Synthesis of 56

[0282] 53 (78 mg, 0.19 mmol) and 15a (80 mg, 0.19 mmol) were reacted asdescribed in example 36, step 1 to give after deprotection with 4-AMP 56as a pale yellow foam, used with no further purification. ¹H NMR (300MHz, DMSO-d₆): δ 8.64 (d, J=8.0 Hz, 1 H) 8.29 (t, J=5.9 Hz, 1 H); 7.43(d, J=7.4 Hz, 2 H); 5.95 (app tt J=4.0, 56.3 Hz, 1 H), 4.34 (m, 1 H);4.02 (m, 1 H); 3.26 (m, 4 H); 2.78 (t, J=6.8 Hz, 2 H); 2.70 (t, J=10.1Hz, 1 H); 2.03 (m, 3 H); 1.79 (m, 2 H); 1.60 (m, 4 H); 1.50 (s, 9 H);1.14 (m, 4 H); 0.85 (m, 2 H). MS (m/z) 558 (M+1)⁺.

Synthesis of 539

[0283] To an ice-cold solution of 56 (100 mg, 0.179 mmol),(L)-Fmoc-Val-OH (67 mg, 0.187 mmol) and HATU (102 mg, 0.268 mmol) in DCM(10 ml) was added DIPEA (62 μl, 0.358 mmol). The solution was stirredfor two days, diluted with EtOAc (100 ml) and washed with 1N HCl,saturated NaHCO₃ and brine (3×50 ml). Drying and evaporation gave thecoupling product (150 mg, 91%). The Fmoc group was cleaved with 4-AMP(0.81 ml, 6.8 mmol) in DCM (2.5 ml) for 3 hours at room temp. Afterdilution of the mixture with EtOAc (50 ml) it was washed with phosphatebuffer (pH 5.5, 6×100 ml) and brine (100 ml). The foam obtained 100 mg(100 mg, 89%) of was used with no further purification. A part of thismaterial (80 mg, 0.122 mmol) in DCM (1 ml) and DIPEA (42.5 μl, 0.183mmol) was treated with a solution of neo-pentyl-chloroformate in DCM (1ml). The solution was stirred overnight, diluted with EtOAc (30 ml) andwashed with 1N HCl, saturated NaHCO₃ and brine (3×20 ml). The crude (90mg) obtained was treated for 3 hours at room temperature withTFA/CH₂Cl₂/H₂O (65:35:5, 15 mL). After removal of the solvents theresidue was freeze-dried from CH₃CN/H₂O 1:1 (50 mL). The powder waspurified by preparative RP-HPLC: Symmetry Waters column (C18, 30×50 mm),flow 40 ml/min, gradient linear from 80% to 38% A over 13 minutes, thenisocratic (RT 14.6 minutes), 48 mg 0 f 539 (57%).

[0284]¹H-NMR (DMSO-d₆) δ 13.5 (bs, 1 H), 8.18 (d, J=8.5 Hz, 1 H), 8.09(t, J=5.8 Hz, 1 H), 7.49 (s, 1 H), 7.48 (s, 1 H), 7.26 (d, J=8.5 Hz, 1H), 5.97 (bt, J=57 Hz, 1 H), 4.30 (m, 2 H), 4.0 (m, 1 H), 3.75 (m, 1 H),3.33-3.21 (m, 4 H), 2.82 (m, 2 H), 2.08-1.90 (m, 5 H), 1.64 (m, 6 H),1.13 (m, 4 H), 0.86 (s, 18 H); MS m/z 715.4 (M+H)⁺.

Example 39 Synthesis of Compounds 551-553

[0285]

Synthesis of 57

[0286] To a solution of the amine hydrogen chloride salt 18 (195 mg,0.665 mmol) in DCM (8 ml) and DIPEA (0.127 ml, 0.731 mmol) at 0° C.under nitrogen were added 21 (152 mg, 0.698 mmol), HOBt (180 mg, 1.33mmol) followed EDC (134 mg, 0.698 mmol) and the solution stirred at roomteperature overnight. The solution was diluted with EtOAc and theorganic phase washed with aqueous HCl (1 N), aqueous saturated NaHCO₃,brine, dried (sodium sulfate) and evaporated in vacuo to leave acolourless glass which was purified by flash chromatography (PE/EtOAc)to afford 227 mg (75%) of the coupling product. ¹H NMR (DMSO-d₆, 400MHz): δ 0.83-0.91 (12 H, m), 1.13-1.33 (7 H, m), 1.51-1.58 (1 H, m),1.64-1.71 (2 H, m), 1.77-1.95 (4 H, m), 2.03-2.11 (1 H, m), 2.15-2.22 (1H, m), 2.77-2.86 (1 H, m), 3.51-3.58 (1 H, m), 3.62-3.76 (3 H, m),3.92-4.09 (5 H, m), 4.33-4.39 (1 H, m), 7.32 (1 H, d, J=8.6 Hz); MS(m/z) 457.1 (M+H)⁺.

[0287] To a solution of the foregoing compound (227 mg, 0.498 mmol) inMeOH (4 ml) was added aqueous NaOH (1 ml, 1 N) and the reaction wasstirred at room temperature for 2 hrs. The solution was diluted withEtOAc and acidified with aqueous HCl (1 N). The organic phase was washedwith brine, dried (sodium sulfate) and evaporated in vacuo to leave 57as a white solid (100%). ¹H NMR (DMSO-d₆, 400 MHz): δ 0.83-0.90 (12 H,m), 1.14-1.34 (5 H, m), 1.51-1.58 (1 H, m), 1.64-1.71 (2 H, m),1.77-1.95 (4 H, m), 2.03-2.10 (1 H, m), 2.15-2.22 (1 H, m), 2.77-2.86 (1H, m), 3.51-3.58 (1 H, m), 3.61-3.76 (3 H, m), 3.91-3.99 (2 H, m),4.26-4.32 (1 H, br dd), 7.31 (1 H, d, J=8.7 Hz); MS (m/z) 429.3 (M+H)⁺.

Synthesis of 58a

[0288] To a solution of the acid 57 (178.5 mg, 0.417 mmol) in DCM (6 ml)at 0° C. under nitrogen were added DIPEA (62.2 mg, 0.481 mmol), 53(181.5 mg, 0.438 mmol), HOBt (112.7 mg, 0.834 mmol) followed EDC (84 mg,0.438 mmol) and the solution stirred at room temperature overnight. Thesolution was diluted with EtOAc and the organic phase washed withaqueous HCl (1 N), aqueous saturated NaHCO₃, brine, dried (sodiumsulfate) and evaporated in vacuo to leave crude 58a (305 mg, 93%) as awhite solid. ¹H NMR (DMSO-d₆, 400 MHz): δ 0.84-0.86 (12 H, br d),1.20-1.37 (5 H, m), 1.53 (10 H, br s), 1.66-1.68(2 H, m), 1.77-1.92 (5H, m), 2.05-2.18 (3 H, m), 2.78-2.83 (3 H, m), 3.20-3.35 (3 H, m),3.56-3.65 (2 H, m), 3.66-3.75 (2 H, m), 3.89-3.94 (2 H, m), 4.25-4.30 (1H, m), 4.34-4.37 (1 H, m), 6.01 (1 H, br t, J_(HF)=57.5 Hz), 7.32 (1 H,d, J=8.7 Hz), 7.45 (2 H, d, J=7.2 Hz), 8.09 (1 H, br t), 8.31 (1 H, d,J=8.6 Hz); MS (m/z) 789.4 (MH)⁺.

Synthesis of 551

[0289] A solution of compound 58a (70 mg, 0.089 mmol) in TFA/DCM/H₂O (6ml/3.5 ml/0.5 ml) was stirred for 2 hrs at room temperature. The solventwas removed in vacuo to leave a glass which was purified by reversephase HPLC (Hibar LiChrosorb RP-18; gradient: 60% A, 2 min isocratic,then in 13 min to 30% A, 5 min isocratic, in 3 min to 10% A). Fractionscontaining the desired product were pooled and lyophilized to givecompound 551. ¹H NMR (DMSO-d₆, 400 MHz): δ 0.83-0.89 (12 H, br d),1.25-1.31 (5 H, m), 1.48-1.54 (1 H, m)), 1.63-1.69 (2 H, m), 1.84-2.20(6 H, m)), 2.79-2.88 (5 H, m), 3.52-3.58 (3 H, m), 3.69-3.75 (3 H, m),3.89-4.00 (2 H, m), 4.25-4.31 (1 H, m), 4.36-4.42 (1 H, m), 5.96 (1 H,br t, J_(HF)=56.0 Hz), 6.76 (1 H, br s), 7.44 (2 H, br d, J=7.0 Hz),7.83 (1 H, br t), 8.01 (1 H, br d); MS (m/z) 733.4 (M+H)⁺.

Synthesis of 552

[0290] To a solution of compound 58a (111 mg, 0.141 mmol) in CHCl₃ (2ml) at 0° C. under nitrogen, mCPBA (technical grade) (32.4 mg, 0.141mmol) was added and the solution stirred at room temperature for 1 hr.The solution was washed with aqueous saturated NaHCO₃, brine, dried(sodium sulfate) and evaporated in vacuo to leave a diastereomericmixture of sulfoxides 58b as a colorless glass, which were immediatelydissolved in a mixture of TFA/DCM/H₂O (6.5 ml/3.5 ml/0.5 ml) and stirredat room temperature for 2 hrs. The solvent was removed in vacuo to leavea white residue which was purified by prepaartive RP-HPLC (HibarLiChrosorb RP-18; gradient: 60% A, 2 min isocratic, then in 13 min to30% A, 5 min isocratic, in 3 min to 10% A). Fractions containing thedesired product were pooled and lyophilized to give compound 552 as amixture of diastereoisomers (1:1*). ¹H NMR (DMSO-d₆, 400 MHz): δ0.82-0.89 (12 H, bd), 1.15-1.21 (1 H, m), 1.30-1.39 (4 H, m), 1.60-1.63(1 H, m), 1.74-2.20 (10 H, m), 2.64-2.70 (1 H, m), 2.79-2.83 (2 H, m),3.18-3.25 (1 H, m), 3.30-3.39 (1 H, m), 3.59-3.79 (4 H, m), 3.87-4.06(10 H, m), 4.24-4.34 (1 H, m), 4.35-4.46 (1 H, m), 5.99 (1 H, br t,J_(HF)=56.5 Hz), 7.25, 7.37*, (1 H, d, J=8.12, 8.4* Hz), 7.49 (2 H, d,J=7.1 Hz), 8.13-8.19 (1 H, m), 8.35, 8.44* (1 H, d, J=8.8, 8.3* Hz); MS(m/z) 749.6 (M+H)⁺.

Synthesis of 553

[0291] To a solution of compound 58a (98 mg, 0.124 mmol) in CHCl₃ (2 ml)at 0° C. under nitrogen, mCPBA (technical grade) (45.04 mg, 0.261 mmol)was added and the solution was stirred at room temperature for 1 hr. Thesolution was washed with aqueous saturated NaHCO₃, brine, dried andevaporated in vacuo to leave 58c as a colourless glass (13) which wasdissolved in a mixture of TFA/DCM/H₂O (6.5 ml/3.5 ml/0.5 ml) and stirredat room temperature for 2 hrs. The solvent was removed in vacuo to leavea white residue which was purified by preparative RP-HPLC (HibarLiChrosorb RP-18; gradient: 60% A, 2 min isocratic, then in 13 min to30% A, 5 min isocratic, in 3 min to 10% A). Fractions containing thedesired product were pooled and lyophilized to give compound 553. ¹H NMR(DMSO-d₆, 400 MHz): δ 0.82-0.86 (12 H, br d), 1.12-1.20 (1 H, m),1.28-1.40 (4 H, m), 1.61-1.64 (1 H, m), 1.76-2.24 (10 H, m), 2.82 (2 H,br t), 3.18-3.36 (3 H, m), 3.66-4.15 (6 H, m), 4.26-4.30 (1 H, m),4.44-4.48 (1 H, m), 6.00 (1 H, br t, J_(HF)=54.5 Hz), 7.35 (1 H, d,J=8.4 Hz), 7.49 (2 H, d, J=7.4 Hz), 8.14 (1 H, br t), 8.44 (1 H, d,J=8.3 Hz); MS (m/z) 765.4 (M+H)⁺.

Example 40 Synthesis of Compounds 555

[0292]

Synthesis of 59

[0293] The synthesis proceeds analogous to example 36, with the couplingof 14b (71 mg, 0.30 mmol) to 9 (74 mg, 0.33 mol) using EDC (68 mg, 0.36mmol) and HOBt (55 mg, 0.36 mmol) in DCM/DMF (4 mL, 7:1). The reactionmixture was stirred overnight, and the crude product obtained afterworkup was dissolved in EtOAc (2 mL). A solution of hydrogen chloride inEtOAc (3.9 M, 2 mL) was added. The mixture was left at room temperaturefor 1 h and then evaporated to give 58 as a colorless solid (114 mg,quantitative).

Synthesis of 60

[0294] The coupling to 15b was performed as described above using 15b(41 mg, 0.14 mmol) and 59 (60 mg, 0.16 mmol) in the presence of EDC (32mg, 0.17 mmol), HOBt (26 mg, 0.17 mmol) and DIPEA (0.037 mL, 0.21 mmol)in DCM (1.5 mL). After deprotection as described above the aminehydrochloride 59 (66 mg, 90%) was obtained.

Synthesis of 555

[0295] 60 (66 mg) was added to a stirred solution of 21 (29 mg, 0.13mmol), HATU (57 mg, 0.15 mmol) and 2,6-lutidine (0.081 mL, 0.44 mmol) inDMF (1 mL). After 3 h at room temperature the reaction was diluted withEtOAc, washed with HCl (1 N), saturated aqueous sodium bicarbonate andbrine. Preparative RP-HPLC (Prep NOVAPAK (Waters) C18 cartridge column,7 micron, 25×100 mm; flow: 10 mL/min; gradient: 50% A, 2 min isocratic,then in 12 min to 20% A; RT 11.0 min) gave 18 mg of 555. ¹H NMR (300MHz, DMSO-d₆): δ 8.18 (d, J=8.2 Hz, 1 H); 8.11 (m, 1 H); 7.66 (d, J=7.0Hz, 2 H); 7.25 (d, J=8.4 Hz, 1 H); 5.97 (m, 1 H); 4.31 (m, 2 H); 3.98(m, 1 H); 3.35 (m, 1 H); 3.26 (m, 2 H); 2.83 (m, 2 H); 2.05 (m, 7 H);1.89 (m, 2 H); 1.78 (m, 1 H); 1.62 (m, 6 H); 1.10 (m, 4 H); 0.85 (m, 14H). MS (m/z) 725 (M+1)⁺.

Example 41 Synthesis of Compound 566

[0296]

Synthesis of 61

[0297] As described in example 36, 14a (106 mg, 0.39 mmol) was coupledto 12 (160 mg, 0.39 mmol) using EDC (82 mg, 0.43 mmol) and HOBT (66 mg,0.43 mmol) in DCM (3 mL). After workup the crude product was purified byflash chromatography (PE/EtOAC 3:1) to afford 110 mg of the couplingproduct, a part (96 mg, 0.19 mmol) of which was dissolved in methanol (5mL) containing Pd/C (10 mg, 10% Pd). The reaction was stirred under anatmosphere of hydrogen for 16 h. Filtration and evaporation gave 61 as acolorless oil (68 mg, 95%). MS (m/z) 381 (M+H)⁺.

Synthesis of 62

[0298] The foregoing compound (63 mg, 0.17 mmol) was coupled to 15b (29mg, 0.19 mmol) analogous to the procedure described in example 36, step2. After workup, the crude product was treated with a solution ofhydrogen cloride in EtOAc (3 M, 2 mL) for 3 h at room temperature.Evaporation gave 62 as a yellow foam (105 mg, quantitative). ¹H NMR (400MHz, DMSO-d₆): δ 9.58 (bs, 1 H), 8.87 (d, J=8.0 Hz, 1 H), 8.50 (bs, 1H); 8.38 (d, J=6.8 Hz, 1 H), 6.93 (app. d, J=7.3 Hz, 2 H), 6.03 (bt,J=56.3 Hz, 1 H), 4.95 (q, J=6.7 Hz, 1 H), 4.38 (m, 1 H), 4.21 (m, 1 H),3.67 (s, 3 H), 3.19 (m, 2 H); 2.82 (m, 2 H), 2.76 (m, 2 H); 2.15 (m, 1H); 2.04 (m, 1 H); 1.89 (m, 3 H); 1.64 (m, 5 H); 1.49 (d, J=6.7 Hz, 3H), 1.19 (m, 4 H); 0.85 (m, 2 H). MS (M/Z) 725 (M+1) MS (M/Z) 745 (M+H).MS (M/Z) 560 ((M+H)⁺, free amine).

Synthesis of 566

[0299] To compound 62 (59 mg, 0.1 mmol) was coupled 21 (24 mg, 0.11mmol) as described in example 36, step 3. The crude product obtainedafter work-up was dissolved in methanol (2 mL). Sodium hydroxide (1 N, 1mL) was added and the reaction stirred at room temperature for 20 min.The solution was diluted with water and acetonitrile and 566 isolated bypreparative RP-HPLC. Column: Waters NovaPak HR C18 cartridge (60 micron,25×100 mm); flow 10 mL/min; gradient 50% A, 1 min isocratic, in 12 minto 15% A. 32 mg of a colorless powder (RT 12.9 min) was obtained afterlyophilization. ¹H NMR (2, diastereomers, 1:1; 400 MHz, DMSO-d₆): δ 13.0(bs, 1 H), 8.20 (d, J=6.8 Hz, 1 H), 8.11 (bs, 1 H); 7.26 (d, J=8.5 Hz, 1H), 6.92 (m, 2 H), 5.98 (bt, J=54.1 Hz, 1 H), 4.80 (bs, 1 H), 4.32 (m, 2H), 4.00 (m, 1 H), 3.74 (m, 3 H), 3.28 (m, 2 H); 3.16 (m, 1 H), 2.74 (m,2 H); 2.08 (m, 4 H); 1.91 (m, 2 H); 1.80 (m, 1 H); 1.64 (m, 5 H); 1.48(d, J=6.7 Hz, 3 H), 1.13 (m, 4 H); 0.85 (m, 14 H); MS (m/z) 745.5(M+H)⁺.

Example 42 Synthesis of Compound 616

[0300]

[0301] A solution of 53 (44 mg, 0.12 mmol), 20 (50 mg, 0.1 mmol) andHATU (60 mg, 0.16 mmol) in DCM (1 mL) and DIPEA (0.037 mL, 0.2 mmol) wasstirred at room temperature overnight. The solid obtained after workupfrom EtOAc was deprotected using 4-AMP as described in example 36. Crude63 (44 mg, 72%) was used without further purification. MS (m/z) 610(M+H)⁺.

[0302] To a stirred solution of 63 (24 mg, 0.04 mmol) and HATU (18 mg,0.048 mmol) in DCM/DMF (1:1, 1 mL) was added DIPEA (0.015 mL, 0.09mmol). Workup after 2 d gave an orange oil (25 mg), which wasdeprotected with TFA/DCM/H₂O (65:30:5, 1 mL) for 30 min at roomtemperature. The crude product obtained after evaporation of thesolvents was purified by preparative RP-HPLC (gradient: 70% A, 3 minisocratic, in 15 min to 10% A; 8 mg, 31%). ¹H-NMR (400 MHz, DMSO-d₆,10:1 mixture of rotamers) δ 13.4 (bs, 1 H), 8.33 (d, J=8.8 Hz, 1 H),8.09 (t, J=6.2 Hz, 1 H), 7.46 (app. d, J=7.9 Hz, 2 H), 7.23 (m, 2 H),7.15 (m, 3 H), 6.01 (bt, J=56.4 Hz, 1 H), 4.30 (m, 2 H), 4.06 (bs, 1 H),3.87 (d, J=5.5 Hz, 1 H), 3.73 (d, J=11.4 Hz, 1 H), 3.49 (dd, J=3.9, 11.4Hz, 1 H), 3.35 (m, 2 H), 3.19 (m, 2 H), 2.79 (t, J=6.8 Hz, 2 H), 2.57(d, J=7.6 Hz, 2 H), 2.12 (m, 2 H), 2.00 (m, 1 H); 1.70-1.87 (m, 4 H),0.83 (d, J=6.7 Hz, 3 H), 0.75 (d, J=6.7 Hz, 3 H). MS (m/z) 654.4 (M+H)⁺.

Example 43 Synthesis of Compound 636

[0303]

[0304] The dipeptide 54 (30 mg, 0.054 mmol) was dissolved inDCM/DMF(1:1, 2 mL). (S)-hexahydromandelic acid (9.5 mg, 0.060 mmol) andHATU (23 mg, 0.060 mmol) were added at 0° C., followed by 2,6-lutidine(14 μl, 0.12 mmol). The cooling bath was removed and the resultingsolution stirred overnight After dilution with EtOAc the organic phasewas washed with aqueous hydrochloric acid (1 N, 2×), saturated aqueoussodium bicarbonate (2×) and brine. Drying over sodium sulfate andevaporation gave a solid, which was purified by Biotage (column 12×7.5;PE/EtOAc 7:3). MS (m/z) 692 (M+H)⁺.

[0305] The foregoing compound (24 mg, 0.034 mmol) was treated withTFA/DCM/H2O (65:35:5, 1 mL), for 30 min at room temperature. Thesolvents were removed under vacuum, taken up with Et₂O and concentratedto give a white solid.

[0306]¹H NMR (DMSO-d₆): δ 8.25 (d, J=8.35 Hz, 1 H), 8.10 (t, J=5.83 Hz,1 H), 7.45 (d, J=7.37 Hz, 2 H), 7.24 (m, 5 H), 5.96 (tt, J=56.5, 4.7 Hz,1 H), 4.43 (t, J=6.55 Hz, 1 H), 4.30 (m, 1 H), 3.97 (dd, J=8.53, 6.07Hz, 1 H), 3.86 (d, J=6.20 Hz, 1 H), 3.57 (m, 2H), 3.38 (m, 2 H, underwater), 3.19 (m, 2H), 2.79 (t, 6.54 Hz, 2 H), 2.16 (m, 2H), 2.09 (m, 1H), 1.60 (m, 6 H), 1.06 (m, 4H). MS (m/z) 636 (M+H)⁺.

Example 44 Synthesis of Compound 708a,b

[0307]

Synthesis of 64

[0308] To a solution of acid 25 (755 mg, 1.643 mmol), (L)-Leu-OBntosylate (776 mg, 1.971 mmol) and HATU (687 mg, 1.807 mmol) in DCM (150ml) cooled to 0° C. was added DIPEA (0.53 ml, 4.11 mmol). The coolingbath was removed and the mixture stirred at room temperature for twodays. The reaction mixture was diluted with EtOAc (400 ml), washed with1 N aqueous HCl, saturated aqueous NaHCO₃ and brine. Drying andevaporation gave the coupling product as a mixture of two diastereomers(1.10 g, 100%). MS (m/z) 663.8 (M+H)⁺.

[0309] To a solution of above compound (1.09 g, 1.6 mmol) in MeOH (75ml) was added Pd/C 30% (230 mg). The reaction mixture was stirred atroom temperature under hydrogen (atmospheric pressure) for 18 hours.Dilution with EtOAc, filtration and evaporation of the solvents gaveindoline 64 in quantitative yield. ¹H-NMR (DMSO-d₆) δ 7.75 (d, J=Hz, 1H), 7.60 (d, J=Hz, 1 H), 7.50 (bs, 2 H), 7.4 (d, J=Hz, 2 H), 7.35 (d,J=Hz, 2 H), 7.0 (m, 2 H), 6.81 (m, 2 H), 6.70 (m, 2 H), 6.52-6.60 (m, 2H), 4.32 (m, 2 H), 4.11 (d, J=14.2 Hz, 2 H), 3.5-3.6 (m, 2 H), 3.15-3.30(m, 2 H), 1.55-1.80 (m, 6 H), 1.50 (s, 36 H), 0.85 (s, 18 H); MS (m/z)573.7 (M+H)⁺.

[0310] To a solution of 64 (138 mg, 0.241 mmol), 52 (105 mg, 0.253 mmol)and, HOBt (34.2 mg, 0.253 mmol) in DCM (5.0 ml) cooled to 0° C. wasadded DIPEA (0.088 ml, 0.506 mmol) and EDC (48.5 mg, 0.253 mmol). Afteraddition the cooling bath was removed and the mixture stirred at roomtemperature for 18 hours. The reaction mixture was diluted with EtOAc(25 ml), washed with 1 N aqueous HCl, saturated aqueous NaHCO₃ andbrine. Drying and evaporation gave a yellow foam which was purified byflash chromatography on silica gel (PE/EtOAc 3:1, Et₃N 0.1%). 115 mg(50%) of the coupling product were obtained. MS (m/z) 951.1 (M+H)⁺.

Synthesis of 708a,b

[0311] The above compound (115 mg) was treated for 3 hours at room temp.with TFA/CH₂Cl₂/H₂O (65:35:5, 40 mL). After removal of the solvents theresidue was freeze-dried from CH3CN/H₂O 1:1 (50 mL) obtaining the TFAsalt of crude 708 as a mixture of 2 diastereomers (95 mg, 94%).

[0312] Purification and separation of the isomers was achieved bypreparative RP-HPLC: Gradient linear from 70% to 37% A over 17 min, thenisocratic. The first fraction (RT=10.0 min) is diastereomer 708a. ¹H-NMR(DMSO-d₆) δ, 7.96 (d, J=8.1 Hz, 1 H), 7.89 (t, J=6.2 Hz, 1 H), 7.66 (d,J=7.8 Hz, 1 H), 7.50 (d, J=5.2 Hz, 1 H), 7.46 (s, 1 H), 7.44 (s, 1 H),6.98 (d, J=5.2 Hz, 1 H), 6.84 (m, 2 H), 6.48 (m, 2 H), 5.84 (t, J=57.3Hz, 1 H), 4.27 (m, 1 H), 4.22 (m, 1 H), 3.67 (d, J=14.2 Hz, 1 H),3.35-3.03 (m, 5 H), 2.78 (t, J=7 Hz, 2 H), 2.08 (m, 2 H), 1.44 (m, 3 H),0.82 (m, 6 H). The second peak corresponds to 708b (RT=10.8 min), 38 mgafter lyophylization. ¹H-NMR (DMSO-d₆) δ, 8.02 (d, J=8.1 Hz, 1 H), 7.96(t, J=6.2 Hz, 1 H), 7.72 (d, J=7.8 Hz, 1 H), 7.46 (s, 2 H), 7.45 (d,J=5.2 Hz, 1 H), 7.0 (d, J=5.2 Hz, 1 H), 6.82 (m, 2 H), 6.47 (m, 2 H),5.91 (t, J=57.3 Hz, 1 H), 4.34 (m, 1 H), 4.25 (m, 1 H), 3.67 (d, J=14.2Hz, 1 H), 3.4-3.15 (m, 3 H), 3.1 (s, 2 H), 2.8 (t, J=7 Hz, 2 H), 2.11(m, 2 H), 1.41 (m, 3 H), 0.76 (m, 6 H). MS (m/z) 721.5 (M+H)⁺.

Example 44 Synthesis of Compound 713

[0313]

Synthesis of 65

[0314] A solution 27 (989 mg) in MeOH (60 mL) was treated with Pd/C(10%, 200 mg) and was stirred under an atmosphere of hydrogen for 15 h.The mixture was filtered and concentrated and the residue (600 mg) wastaken up in DCM (3 mL) and then treated with L-leucine methylester(291.9 mg, 1.6 mmol) and DIPEA (0.84 mL, 4.8 mmol). The solution wascooled to 0° C. and HATU (611 mg, 1.61 mmol) was added. The mixture wasstirred for 6 h and then diluted with hydrochloric acid (1 N) andextracted with EtOAc. The organic layer was washed with NaHCO₃ and brineand then dried over Na₂SO₄. Concentration afforded a residue which waspurified by chromatography (biotage 40×70, 90:10 petroleum ether:ethylacetate eluent) to give an oil (489 mg), a portion of which (450 mg) wastaken up in MeOH (5 mL) and treated dropwise with a 1 N NaOH(aq) (1.1mL). The mixture was stirred for 8 h at room temperature, then acidifiedand extracted with EtOAc. Concentration of the dried organic layer gave65 as a mixture of diastereoisomers (1:1). ¹H NMR (DMSO-d₆) 7.73 (m, 1H), 7.62 (app. t, J=5.10, 1 H), 7.14 and 7.07 (d, J=5.10 Hz, 1 H), 6.83(m, 1 H), 6.75 (m, 1 H), 6.50 (d, J=7.97 Hz, 1 H), 6.40 (m, 1 H), 5.88and 5.66 (s, 1 H), 4.21 (m, 1 H), 3.69-3.57 (m, 1 H), 3.22-3.13 (m, 1H), 2.47-2.33 (m, 3 H), 2.27 (m, 1 H), 1.49 and 1.47 (s, 9 H), 1.42 (m,2 H), 1.19 (m, 1 H), 0.81 (m, 3 H), 0.62 (m, 3 H).

Synthesis of 713

[0315] A solution of 65 (95 mg, 0.195 mmol) in DCM (5 mL) was treatedwith DIPEA (0.034 mL, 0.195 mmol), HOBt (26.4 mg, 0.195 mmol), and 53(81 mg, 0.195 mmol). The solution was cooled to 0° C. and EDC (37.4 mg,0.195 mmol) was added. After stirring for 15 h at room temperature, themixture was diluted with hydrochloric acid and extracted with EtOAc.Concentration of the dried organic layer afforded a residue which wastreated with a 60/10/30 mixture of TFA/H2O/DCM (10 mL) and stirred for 1h at room temperature. The solution was concentrated and the solid waspurified by HPLC (Column: Nucleosil C18 (Machery Nagel), 25×2.5 cm; 45%A isocratic) to give 713 as the second fraction.

[0316] First diastereomer: 14 mg (10%); ¹H NMR (DMSO-d₆) 8.14 (dd,J=5.8, 6.0 Hz, 1 H), 8.10 (d, J=8.2 Hz, 1 H), 7.66 (m, 2 H), 7.49 (m, 2H), 7.11 (d, J=5.1 Hz, 1 H), 6.87 (m, 1 H), 6.76 (d, J=7.3 Hz, 1 H),6.52 (d, J=7.9 Hz, 1 H), 6.42 (m, 1 H), 5.91 (bs 1 H), 5.84 (bt, J=56.5Hz, 1 H), 4.25 (m, 2 H), 3.69 (d, J=13.3 Hz, 1 H), 3.20 (d, J=13.3 Hz, 1H), 3.18 (m, 2 H), 2.80 (t, J=6.5 Hz, 2 H), 2.37 (m, 4 H), 2.00 (m, 2H), 1.43 (m, 2 H), 1.11 (m, 1 H), 0.86 (d, J=6.8 Hz, 3 H), 0.82 (d,J=7.2 Hz, 3 H); MS (m/z) 735.4 (M+H)⁺. 713: 14 mg (10%); ¹H NMR(DMSO-d₆) 8.22 (t, J=8.2 Hz, 1 H), 8.19 (d, J=5.1 Hz, 1 H), 7.67 (d,J=7.9 Hz, 1 H), 7.61 (d, J=5.1 Hz, 1 H), 7.49 (m, 2 H), 7.10 (d, J=5.1Hz, 1 H), 6.87 (m, 1 H), 6.78 (d, J=7.5 Hz, 1 H), 6.54 (d, J=7.8 Hz, 1H), 6.44 (m, 1 H), 6.07 (bs, 1 H), 5.95 (bt, J=56.2 Hz, 1 H), 4.35 (m, 1H), 4.23 (m, 1 H), 3.62 (d, J=13.4 Hz, 1 H), 3.25 (d, J=13.4 Hz, 1 H),3.20 (m, 2 H), 2.81 (t, J=6.8 Hz, 2 H), 2.50-2.25 (m, 3 H), 2.18-2.01(m, 2 H), 1.34 (m, 2 H), 1.16 (m, 2 H), 0.68 (d, J=6.6 Hz, 3 H), 0.65(d, J=6.5 Hz, 3 H); MS (m/z) 735.6 (M+H)⁺.

Example 45 Synthesis of Compound 576

[0317]

[0318] The title compound was prepared using the procedure described inExample 36 by coupling compound 53 with(4R)-1-(tert-butoxycarbonyl)-4-[(7-methoxy-2-phenylquinolin-4-yl)oxy-proline(prepared as described in WO 0009543, example 7). After coupling andwork-up, the crude product was treated with TFA to remove the protectinggroups, and then coupled to Boc-tert-butyl glycine to give the finalproduct after purification by RP-HPLC.

[0319]¹H NMR (400 MHz, DMSO-d₆): δ 8.45 (d, J=8.4 Hz, 1 H); 8.25-8.15(m, 4 H); 7.20-7.05 (m, 4 H); 7.05-7.45 (m, 3H); 7.25-7.15 (m, 1H); 6.78(d, J=7.8 Hz, 1 H); 6.13 (app dt J=4.3, 56.0 Hz, 1 H), 5.77 (bs, 1 H);4.60-4.50 (m, 2 H); 4.40-4.25 (m, 1H); 4.02 (d, J=8.0 Hz, 1 H); 3.96 (s,3H); 3.89 (d, J=10.8 Hz, 1 H); (peaks overlapped by H₂O bs); 3.25-3.15(m, 2 H); 2.30-1.95 (m, 3H); 1.22 (s, 9 H); 0.96 (s, 9 H). MS (m/z) 882+(M+1)⁺.

Inhibition of NS3 Protease Example 46 Full-Length NS3-NS4A HeterodimerProtein Assay (Assay A)

[0320] (Gallinari, P., Paolini, C., Brennan, D., Nardi, C., Steinkühler,C. and De Francesco, R. Modulation of hepatitis C virus NS3 protease andhelicase activities through the interaction with NS4A. Biochemistry 38,5620-5632, 1999)

[0321] In this assay the full length NS3 protein spanning residues1027-1657 of the HCV polyprotein, noncovalently complexed with thefull-length NS4A cofactor protein (residues 1658-1711) was used. Assayswere performed in 60 μl of a buffer containing 50 mM Hepes pH 7.5, 0.15M NaCl, 0.1% (v/v) Triton X-100, 15% (v/v) glycerol, 10 mM DTT, 2 nMNS3/NS4A complex. To this assay mix 10 μM (final concentration) of apeptide substrate corresponding to the NS5AB cleavage site and havingthe sequence H-EAGDDIVPCSMSYTWTGA-OH, were added. Incubation times at23° C. were adjusted to obtain <10% conversion. Reactions were stoppedby the addition of 40 μl of 1% TFA. Cleavage was determined by HPLCusing a Merck-Hitachi chromatograph equipped with an autosampler. 80 μlsamples were injected on a Lichrospher C18 reversed phase cartridgecolumn (4×74 mm, 5 μm, Merck) and fragments were separated using a10-40% acetonitrile gradient at 5%/min using a flow rate of 2.5 ml/min.Peak detection was done by monitoring both the absorbance at 220 nm andtryptophan fluorescence (λ_(ex)=280 nm, λ_(em)=350 nm). Cleavageproducts were quantitated by integration of chromatograms with respectto appropriate standards. Kinetic parameters were calculated fromnonlinear least-squares fit of initial rates as a function of substrateconcentration with the help of a Kaleidagraph software, assumingMichaelis-Menten kinetics.

[0322] K_(i) values of peptide inhibitors were calculated from substratetitration experiments performed in the presence of increasing amounts ofinhibitor. Experimental data sets were simultaneously fitted to eg.1using a multicurve fit macro with the help of a Sigmaplot software:

V=(V _(max) S)/(K _(m)(1+K _(i) /I)+S);   (eq.1)

[0323] Alternatively, K_(i) values were derived from IC50 values,calculated using a four-parameter logistic function, according to eq.2:

IC50=(1+S/K _(m))K _(i)   (eq.2)

Example 47 HCV NS3 Protease Domain/ NS4A Cofactor Peptide Assay (AssayB)

[0324] (Steinkühler, C., Biasiol, G., Brunetti, M., Urbani, A., Koch,U., Cortese, R., Pessi, A., and De Francesco, R. Product inhibition ofthe hepatitis C virus NS3 protease. Biochemistry 37, 8899-8905, 1998)

[0325] In this assay the NS3 protease domain (residues 1027-1206 of theHCV polyprotein) complexed to a modified form of the NS4A peptide,spanning residues 21-34 of the NS4A cofactor in addition to asolubilizing lys-tag, Pep 4AK [KKKGSVVIVGRIILSGR(NH₂)] were used. Assubstrate, peptide 4AB [DEMEECASHLPYK] based on the sequence of theNS4A/NS4B cleavage site of the HCV polyprotein, was added.

[0326] Cleavage assays were performed in 60 μl 50 mM Hepes pH7.5, 1%(w/v) CHAPS, 15% (v/v) glycerol, 10 mM DTT (buffer A), containing 80 μMPep4AK. Buffer solutions were preincubated for 10 min with 10-200 nMprotease and reactions were started by addition of 10 μM substrate. Sixduplicate data points at different substrate concentrations were used tocalculate kinetic parameters. Incubation times were chosen in order toobtain <7% substrate conversion and reactions were stopped by additionof 40 μl 1% TFA. Reactions were analysed by HPLC essentially asdescribed above. The fluorescence detector was set to monitor tyrosinefluorescence of substrate and cleavage product (λ_(ex)=260 nm,λ_(em)=305 nm).

Tables of Compounds

[0327] The following tables list compounds representative of theinvention. All compounds were characterized by ¹H-NMR and massspectroscopy. Compounds of the invention were either assayed in one orboth of the assays described in examples 46 and 47 and were found to beactive with IC₅₀ below 100 μM (C) below 10 μM (B) or below 1 μM (A). Alist of abbreviations used in the tables can be found at the beginningof the Examples. TABLE 1

Assay No. R₅ R₄ R₂ R₁ A Assay B 101 Cbz

—CH₂—SH

C 102

—CH₂—SH

C 103 Boc

—CH₂—SH

C 104 i-Boc

—CH₂—SH

C C 105 Boc

—CH₂—CHF₂

C C 106 i-Boc

—CH₂—SH

C 107 i-Boc

—CH₂—SH

C 108 i-Boc

—CH₂—SH

C 109 i-Boc

—CH₂—SH

C 110 i-Boc

—CH₂—SH

C 111 i-Boc

—CH₂—SH

C 112 i-Boc

—CH₂—SH

C 113 i-Boc

—CH₂—SH

C 114 Boc

—CH₂—SH

C B 115 Boc

—CH₂—CHF₂

C B 116 Cbz

—CH₂—SH

C 117 i-Boc

—CH₂—SH

C 118 i-Boc

—CH₂—SH

C 119 i-Boc

—CH₂—SH

C 120 i-Boc

—CH₂—SH

C 121 i-Boc

—CH₂—SH

C 122 i-Boc

—CH₂—SH

C 123 i-Boc

—CH₂—SH

C 124 i-Boc

—CH₂—SH

C 125 i-Boc

—CH₂—CHF₂

C 126 i-Boc

—CH₂—SH

C 127 i-Boc

—CH₂—SH

C 128 i-Boc

—CH₂—SH

C 129 i-Boc

—CH₂—SH

C 130 i-Boc

—CH₂—SH

C 131 i-Boc

—CH₂—SH

C 132 i-Boc

—CH₂—SH

C 133 i-Boc

—CH₂—SH

C B 134 i-Boc

—CH₂—SH

B B 135 i-Boc

—CH₂—SH

C C 136 i-Boc

—CH₂—SH

C 137 i-Boc

—CH₂—SH

C 138 i-Boc

—CH₂—SH

C 139 i-Boc

—CH₂—SH

C 140 i-Boc

—CH₂—SH

C 141 i-Boc

—CH₂—SH

C 142 Boc

—CH₂—SH

C 143 Cbz

—CH₂—SH

C 144 i-Boc

—CH₂—SH

C 145 i-Boc

—CH₂—SH

C 146 i-Boc

—CH₂—SH

C 147 i-Boc

—CH₂—SH

C 148 i-Boc

—CH₂—SH

C 149 i-Boc

—CH₂—SH

C 150 i-Boc

—CH₂—SH

C 151 i-Boc

—CH₂—SH

C B 152 i-Boc

—CH₂—SH

B B 153 i-Boc

—CH₂—SH

C 154 i-Boc

—CH₂—SH

C B 155 i-Boc

—CH₂—SH

C 156 i-Boc

—CH₂—SH

C B 157 i-Boc

—CH₂—SH

B B 158 i-Boc

—CH₂—SH

B B 159 i-Boc

—CH₂—SH

B 160 i-Boc

—CH₂—SH

B B 161 i-Boc

—CH₂—SH

B 162 i-Boc

—CH₂—SH

B 163 i-Boc

—CH₂—SH

B 164 i-Boc

—CH₂—SH

C B 165 i-Boc

—CH₂—SH

B B 166 i-Boc

—CH₂—CHF₂

C B 167 Boc

—CH₂—SH

C 168 alloc

—CH₂—SH

C 169 Cbz

—CH₂—SH

C 170

—CH₂—SH

C 171

—CH₂—SH

C B 172 i-Boc

—CH₂—SH

C 173 i-Boc

—CH₂—SH

C B 174 i-Boc

—CH₂—SH

C 175 i-Boc

—CH₂—SH

C 176 i-Boc

—CH₂—SH

C 177 i-Boc

—CH₂—SH

C B 178 i-Boc

—CH₂—SH

B A 179 i-Boc

—CH₂—CHF₂

B B 180 i-Boc

—CH2—CHF2

B A 181

—CH₂—SH

B B 182

—CH₂—SH

C 183

—CH₂—SH

B 184

—CH₂—SH

B 185 i-Boc

—CH₂—SH

C 186 i-Boc

—CH₂—SH

A 187 i-Boc

—CH₂—CHF₂

A A 188 i-Boc

—CH₂—SH

B 189 i-Boc

—CH₂—SH

C 190 i-Boc

—CH₂—SH

B 191 i-Boc

—CH₂—SH

C 192 i-Boc

—CH₂—SH

C 193 i-Boc

—CH₂—SH

C

[0328] TABLE 2

No. R₅ R₁ Assay A Assay B 201

C 202

C 203

C C 204

C 205

C 206

C 207

C 208

C 209

C 210

C 211

C 212

C 213

C 214

C 215

C 216

C 217

C 218

C 219

C 220

C 221

B 222

C 223

C 224

C 225

B 226

C 227

C 228

C 229

C 230

C 231

C 232

C 233

C 234

C 235

C 236

C 237

C 238

C 239

C 240

C 241

C 242

B 243

C 244

C 245

C 246

C 247

C 248

C 249

C 250

C 251

C 252

B B 253

C 254

C 255

C 256

C

[0329] TABLE 3

No. R₅ R₂ R₁ Assay A Assay B 301 AcDif —CH₂—CHF₂

C 302 AcDif —CH₂—SH

C 303 AcAspGluDif —CH₂—SH

A 304 AcAspGluDif —CH₂—CHF₂

A 305 AcAspGluDif —CH₂—CHF₂

A 306 AcAspGluDif —CH₂—SH

A 307 AcAspGluDif —CH₂—SH

A 308 AcAspGluDif —CH₂—SH

A 309 AcAspGluDif —CH₂—SH

A 310 AcAspGluDif —CH₂—SH

A 311 AcAspGluDif —CH₂—SH

A 312 AcAspGluDif —CH₂—SH

A 313 AcAspGluDif —CH₂—SH

A 314 AcAspGluDif —CH₂—SH

A 315 AcAspGluDif —CH₂—SH

A 316 AcAspGluDif —CH₂—SH

A 317 AcAspGluDif —CH₂—SH

A 318 AcAspGluDif —CH₂—SH

A 319 AcAspGluDif —CH₂—SH

A 320 AcAspGluDif —CH₂—SH

A 321 AcAspGluDif —CH₂—CHF₂

A A

[0330] TABLE 4

No.

R₂ R₁ Assay A Assay B 401

—CH₂—SH

C 402

—CH₂—SH

C 403

—CH₂—SH

C 404

—CH₂—SH

C 405

—CH₂—SH

C 406

—CH₂—SH

C 407

—CH₂—SH

C 408

—CH₂—SH

C C 409

—CH₂—SH

B 410

—CH₂—SH

C 411

—CH₂—SH

C 412

—CH₂—SH

C 413

—CH₂—SH

C 414

—CH₂—SH

C 415

—CH₂—SH

C 416

—CH₂—SH

C 417

—CH₂—SH

C 418

—CH₂—SH

C 419

—CH₂—SH

C 420

—CH₂—SH

B 421

—CH₂—CHF₂

C 422

—CH₂—SH

C

[0331] TABLE 5

No.

R₂₀ R₂ R₁ Assay A 501

H —CH₂—SH

C 502

H —CH₂—CHF₂

B 503

OH —CH₂—SH

C 504

OH —CH₂—CHF₂

B 505

OCH₂O—(CH₂)₂OCH₃ —CH₂—CHF₂

B 506

Ph —CH₂—SH

B 507

—CH₂—SH

B 508

Ph —CH₂—SH

C 509

—CH₂—CHF₂

C 510

Ph —CH₂—CHF₂

B 511

Ph —CH₂—CHF₂

C 512

Ph —CH₂—SH

C 513

Ph —CH₂—SH

C 514

Ph —CH₂—SH

C 515

Ph —CH₂—SH

C 516

Ph —CH₂—SH

B 517

Ph —CH₂—CHF₂

B 518

Ph —CH₂—CHF₂

B 519

Ph —CH₂—CHF₂

C 520

Ph —CH₂—SH

B 521

Ph —CH₂—SH

A 522

Ph —CH₂—SH

A 523

Ph —CH₂—CHF₂

A 524

Ph —CH₂—CHF₂

C 525

Ph —CH₂—CH₃

C 526

—CH₂—CHF₂

A 527

—CH₂—CHF₂

B 528

—CH₂—CHF₂

A 529

CH₂—CF₃

B 530

CH₂—CF₃

B 531

—CH₂—CHF₂

B 532

—CH₂—CHF₂

A 533

—CH₂—CHF₂

A 534

Ph —CH₂—CHF₂

B 535

—CH₂—CHF₂

A 536

—CH₂—CHF₂

B 537

Ph —CH₂—CHF₂

B 538

—CH₂—CHF₂

A 539

—CH₂—CHF₂

A 540

—CH₂—CHF₂

A 541

—CH₂—CHF₂

A 542

—CH₂—CHF₂

B 543

—CH₂—CHF₃

B 544

—CH₂—CHF₂

B 545

—CH₂—CHF₂

A 546

—CH₂—CHF₂

B 547

—CH₂—CHF₂

A 548

n-PrS —CH₂—CHF₂

A 549

n-PrS(O) —CH₂—CHF₂

B 550

n-PrS(O)₂ —CH₂—CHF₂

A 551

—CH₂—CHF₂

B 552

—CH₂—CHF₂

A 553

—CH₂—CHF₂

A 554

Ph —CH₂—CHF₂

A 555

—CH₂—CHF₂

A 556

—CH₂—CHF₂

A 557

—CH₂—CHF₂

A 558

Ph —CH₂—CHF₂

B 559

Ph —CH₂—CHF₂

C 560

Ph —CH₂—CHF₂

A 561

Ph —CH₂—CHF₂

A 562

Ph —CH₂—CHF₂

C 563

Ph —CH₂—CHF₂

B 564

Ph —CH₂—CHF₂

B 565

—CH₂—CHF₂

A 566

—CH₂—CHF₂

A 567

—CH₂—CHF₂

A 568

—CH₂—CHF₂

A 569

Ph —CH₂—CHF₂

B 570

Ph —CH₂—CHF₂

A 571

Ph —CH₂—CHF₂

B 572

Ph —CH₂—CHF₂

C 573

Ph —CH₂—CHF₂

C 574

Ph —CH₂—CHF₂

C 575

Ph —CH₂—CHF₂

C 576

—CH₂—CHF₂

A

[0332] TABLE 6 No.

R₁₇ 601

Ph 602

Ph 603

Ph 604

Ph 605

Ph 606

Ph 607

Ph 608

Ph 609

Ph 610

Ph 611

Ph 612

Ph 613

Ph 614

615

616

Ph(CH₂)₃O 617

618

619

620

621

622

OtBu 623

624

625

626

627

628

Ph 629

Ph 630

Ph 631

Ph 632

Ph 633

Ph 634

Ph 635

Ph 636

Ph 637

Ph 638

Ph No. R₂ R₁ Assay A 601 —CH₂—CHF₂

C 602 —CH₂—CHF₂

C 603 —CH₂—CHF₂

C 604 —CH₂—CHF₂

C 605 —CH₂—CHF₂

C 606 —CH₂—CHF₂

C 607 —CH₂—CHF₂

C 608 —CH₂—CHF₂

C 609 —CH₂—CHF₂

C 610 —CH₂—CHF₂

C 611 —CH₂—CHF₂

C 612 —CH₂—CHF₂

C 613 —CH₂—CHF₂

C 614 —CH₂—CHF₂

C 615 —CH₂—CHF₂

C 616 —CH₂—CHF₂

C 617 —CH₂—CHF₂

C 618 —CH₂—CHF₂

C 619 —CH₂—CHF₂

C 620 —CH₂—CHF₂

C 621 —CH₂—CHF₂

C 622 —CH₂—CHF₂

C 623 —CH₂—CHF₂

C 624 —CH₂—CHF₂

C 625 —CH₂—CHF₂

C 626 —CH₂—CHF₂

C 627 —CH₂—CHF₂

C 628 —CH₂—CHF₂

C 629 —CH₂—CHF₂

C 630 —CH₂—CHF₂

C 631 —CH₂—CHF₂

C 632 —CH₂—CHF₂

C 633 —CH₂—CHF₂

C 634 —CH₂—CHF₂

C 635 —CH₂—CHF₂

C 636 —CH₂—CHF₂

C 637 —CH₂—CHF₂

C 638 —CH₂—CHF₂

C

[0333] TABLE 7

No. R₁₃ R₂ R₁ Assay A Assay B 701

—CH₂—SH

C 702

—CH₂—SH

C C 703

—CH₂—SH

C 704

—CH₂—SH

C 705

—CH₂—CHF₂

C B 706

—CH₂—CHF₂

C B 707a

—CH₂—CHF₂

C 707b

—CH₂—CHF₂

C 708a

—CH₂—CHF₂

C 708b

—CH₂—CHF₂

C B 709

—CH₂—CHF₂

C C 710

—CH₂—CHF₂

C B 711a

—CH₂—CHF₂

C 711b

—CH₂—CHF₂

C B 712a

—CH₂—CHF₂

C 712b

—CH₂—CHF₂

C B 713

—CH₂—CHF₂

C C 714

—CH₂—CHF₂

C C 715

—CH₂—CHF₂

C C 716

—CH₂—SH

C C 717

—CH₂—SH

C C 718

—CH₂—CHF₂

C C 719

—CH₂—CHF₂

C C 720

—CH₂—CHF₂

C C 721

—CH₂—CHF₂

C C

1. A compound of Formula (I), or a pharmaceutically acceptable salt orester thereof:

wherein Q is selected from the group consisting of:

wherein X is —CH₂— or —O—; Y is a group of formula —C(R^(a))₂— whereeach R^(a) is independently selected from hydrogen, hydroxyl, carboxylicacid, lower alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl, or thetwo R^(a) groups together form a cycloalkyl group containing 3 to 7carbon atoms; Z is a substituted or unsubstituted aryl or heteroarylgroup; R is a lower alkyl group, optionally substituted with one or morefluorine atoms, or is —CH₂SH; R³ is an optionally substituted alkyl,aryl, heteroaryl, aralkyl, or heteroaralkyl group containing from 2 to16 carbon atoms, or together with R^(c) forms a ring including IS thenitrogen atom which bears R^(c); R^(c) is hydrogen or a lower alkylgroup or together with R³ forms a ring; R⁴ is an alkyl, alkenyl,aralkyl, heteroaralkyl, aryl or heteroaryl group containing from 2 to 16carbon atoms or is an acidic group; R⁵ is selected from (R⁶)₂NCO—,R⁷CO—, R⁷OCO—, R⁷NHCO—, R⁷CO.CO—, R⁷S(O)₂— and R⁸ pep where “pep” is anamino acid, di- or tri-peptide; each R⁶, independently, is selected fromhydrogen and optionally substituted, optionally interrupted lower alkylor lower alkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl groups, orthe two R⁶ taken together form a four to seven membered ring optionallycontaining one or more other heteroatoms in addition to the nitrogenatoms to which the R⁶ groups are bonded; R⁷ is an optionallysubstituted, optionally interrupted alkyl, alkenyl, aralkyl,heteroaralkyl, aryl or heteroaryl group containing from 1-18 carbonatoms; R⁸ is a group of formula (R⁶)₂NCO—, R⁷CO—, R⁷OCO—, R⁷NHCO—,R⁷COCO—, and R⁷S(O)₂—; “pep” if present is an amino acid, di, or tripeptide of formula C—B-A; wherein A is selected from naturally andnon-naturally occurring amino acids having a hydrophobic side chaincontaining 1-20 carbon atoms; B may be absent, in which case C will alsobe absent, but if present is selected from naturally or non-naturallyoccurring amino acids having a side chain which includes an acidicfunctionality; C may be absent, either by itself or together with B, butif present may be selected from naturally or non-naturally occurringamino acids containing an acidic functionality; R¹³ is a groupcontaining up to 25 carbon atoms, 0-5 oxygen atoms, 0-3 nitrogen atoms,0-2 sulphur atoms and up to 9 other heteroatoms which may be the same ordifferent; R¹⁷ is hydrogen, a lower alkyl, lower alkenyl, aryl,heteroaryl, aralkyl, heteroaralkyl, hydroxyl, alkoxy, aryloxy, aralkoxy,heteroaralkoxy, thioether, sulfonyl or sulfoxide group; and R¹⁸ is agroup containing up to 25 carbon atoms, 0-5 oxygen atoms, 0-3 nitrogenatoms, 0-2 sulphur atoms and up to 9 other heteroatoms which may be thesame or different.
 2. A compound as claimed in claim 1 represented byFormula (1):

and pharmaceutically acceptable salts and esters thereof.
 3. A compoundas claimed in claim 1 represented by the following formula:

and pharmaceutically acceptable salts and esters thereof.
 4. A compoundas claimed in claim 1 represented by the formula:

and pharmaceutically acceptable salts and esters thereof.
 5. A specificcompound as depicted in any one of Tables 1 to
 7. 6. A compound offormula (I) as defined in claim 1, or a pharmaceutically acceptable saltor ester thereof, for use in therapy.
 7. A pharmaceutical compositioncomprising a compound of formula (I) as defined in claim 1, or apharmaceutically acceptable salt or ester thereof, in association with apharmaceutically acceptable carrier.
 8. The use of a compound of formula(I) as defined in claim 1, or a pharmaceutically acceptable salt orester thereof, for the manufacture of a medicament for the treatment orprevention of hepatitis C or a related condition.
 9. A process for thepreparation of a compound of formula (I) as defined in claim 1, whichcomprises reacting protected form of the P1 amino acid:

with a compound of formula H₂N—X—Y-Z; and subsequently extending towardsthe N-terminus by conventional methods.
 10. A method of inhibiting HCVNS3 protease activity, and/or of treating or preventing hepatitis C or arelated condition, the method involving administering to a subjectsuffering from the condition a therapeutically or prophylacticallyeffective amount of a compound of formula (I) as defined in claim 1, ora pharmaceutically acceptable salt or ester thereof.